S. cerevisiae

Gene, 1997 Oct 1, 198(1-2), 43 - 52
The ER chaperone encoding bipA gene of black Aspergilli is induced by heat shock and unfolded proteins; van Gemeren IA et al.; We describe the cloning and characterisation of the BiP gene homologues of the filamentous fungi Aspergillus niger and Aspergillus awamori . The BiP genes of these black Aspergilli encode an identical protein of 672 amino acids, which has a high homology with the BiP protein from Saccharomyces cerevisiae and contains a putative signal sequence of 38 amino acids . The DNA sequences of the Aspergillus BiP genes diverge in particular in the three intronic sequences and the 5'- and 3'- noncoding regions . Sequences resembling Heat Shock Elements (HSE) and Unfolded Protein Response (UPR) elements, as found in the yeast KAR2 promoter, are present in the 5' non-transcribed regions of both genes . The expression of the A . niger bipA gene is increased by heat shock and tunicamycin treatment.

FEBS Lett, 1997 Oct 20, 416(2), 135 - 8
The different inhibitory domains of the Oct-2 transcription factor have distinct functional activities; Gay RD et al.; The Oct-2 POU family transcription factor contains three distinct regions whose deletion reduces its ability to inhibit transcription via its octamer binding site . Here we show that only one of these inhibitory domains is capable of also inhibiting the activity of activating molecules bound at adjacent sites upstream of a TATA box-containing promoter whereas the other two regions are inactive in this assay . None of the three regions is able to achieve this effect when located upstream of the same promoter containing an initiator motif . The mechanisms of action of these domains and their role in the functioning of the Oct-2 factor are discussed.

Biochem Biophys Res Commun, 1997 Nov 7, 240(1), 228 - 33
The trimerization domain of human heat shock factor 2 is able to interact with nucleoporin p62; Yoshima T et al.; Heat shock factor 2 (HSF2) acquires DNA binding activity during hemin-induced differentiation of human K562 erythroleukemia cells . To investigate the mechanisms responsible for the regulation of HSF2 activity, we searched for proteins that can associate with HSF2 by the yeast two-hybrid system . Nucleoporin p62, a major component of the nuclear pore complex, was cloned from cDNA libraries of K562 cells . We demonstrated physical interaction between HSF2 and p62 both by a glutathione S-transferase (GST) pull-down assay in vitro and by a two-hybrid assay in K562 cells . HSF1 is also able to interact with p62 on a GST pull-down assay, but not on a mammalian two-hybrid system . Furthermore, it was shown that this interaction occurred between the trimerization domain of HSF2 and the C-terminal alpha-helical coiled-coil domain of p62 . These data suggest the possibility that p62 is involved in the activation or regulation of HSF2.

Biochem Biophys Res Commun, 1997 Oct 29, 239(3), 710 - 4
Overproduction of Mpd2p suppresses the lethality of protein disulfide isomerase depletion in a CXXC sequence dependent manner; Tachikawa H et al.; The third multicopy suppressor gene of the PDI1 deletion from Saccharomyces cerevisiae, MPD2, was isolated and characterized . The MPD2 gene encodes a protein with a putative signal sequence, ER retention signal, and a disulfide isomerase active site like sequence . The amino acid sequence around the active site like sequence is similar to the thioredoxin-like domains of PDI and PDI related proteins, although the similarity is comparatively low . A delta-pdi1 strain over-producing Mpd2p showed slow growth and was sensitive to 1 mM dithiothreitol . Mpd2p can be detected in wild type cells and is a glycoprotein . Although the MPD2 gene was not essential for growth, overexpression of the gene partially restored the maturation defect of carboxypeptidase Y caused by the PDI1 deletion . Mutagenesis analysis revealed that Mpd2p can compensate for the loss of PDI with its CXXC sequence.

J Mol Biol, 1997 Oct 17, 273(1), 1 - 6
A mutant form of mitochondrial GrpE suppresses the sorting defect caused by an alteration in the presequence of cytochrome b2; Merlin A et al.; Transport of preproteins across the inner mitochondrial membrane requires the action of the matrix heat shock protein Hsp70 . Together with its co-chaperone mitochondrial GrpE (Mge1), mtHsp70 transiently binds to the inner membrane translocase subunit Tim44 in a nucleotide-regulated manner, forming an ATP-dependent import driving machinery . We report that a mutant form of Mge1 (Mge1-100) is completely absent in mtHsp70-Tim44 complexes, although its ability to interact with soluble mtHsp70 is only partially reduced . While this mge1-100 mutation only partially retards preprotein translocation into the matrix, it exerts a selective effect on sorting of cytochrome b2 to the intermembrane space . A cytochrome b2 with an altered sorting signal, which is only processed to the intermediate stage and mistargeted to the matrix of wild-type mitochondria, is processed to the mature form and correctly targeted to the intermembrane space of mge1-100 mitochondria . These results suggest that (1) Mge1-100 discriminates between soluble and membrane-bound mtHsp70 and (2) the membrane-bound mtHsp70-Mge1 driving system competes with the sorting machinery for translocation of preproteins like cytochrome b2.

J Invertebr Pathol, 1997 Nov, 70(3), 226 - 33
Heat-shock response in a molluscan cell line: characterization of the response and cloning of an inducible HSP70 cDNA; Laursen JR et al.; Sublethal heat-shock of cells of the Bge (Biomphalaria glabrata embryonic) snail cell line resulted in increased or new expression of metabolically labeled polypeptides of approximately 21.5, 41, 70, and 74 kDa molecular mass . Regulation of this response appeared to be at the transcriptional level since a similar protein banding pattern was seen upon SDS-PAGE/fluorographic analysis of polypeptides produced by in vitro translation of total RNA from cells subjected to heat shock . Using a yeast (Saccharomyces cerevisiae) 70-kDa heat-shock protein (HSP70) probe to screen a cDNA library from heat-treated Bge cells, we isolated a full-length cDNA clone encoding a putative Bge HSP70 . The cDNA was 2453 bp in length and contained an open reading frame of 1908 bp encoding a 636-amino-acid polypeptide with calculated molecular mass of 70,740 Da . Comparison of a conserved region of 209 amino acid residues revealed > 80% identity between the deduced amino acid sequence of Bge HSP70 and that of yeast (81%), the human blood fluke Schistosoma mansoni (for which B . glabrata serves as intermediate host) (81%), Drosophila (81%), human (84%), and the marine gastropod Aplysia californica (88%, 90%) . In addition to the extensive sharing of sequence homology, the identification of several eukaryotic HSP70 signature sequences and an N-linked glycosylation site characteristic of cytoplasmic HSPs strongly support the identity of the Bge cDNA as encoding an authentic HSP70 . Results of a Northern blot analysis, using Bge HSP70 clone-specific probes, indicated that gene expression was heat inducible and not constitutively expressed . This is the first reported sequence of an inducible HSP70 from cells originating from a freshwater gastropod and provides a first step in the development of a genetic transformation system for molluscs of medical importance.

J Biol Chem, 1997 Nov 14, 272(46), 29372 - 9
Activation of the Ste20-like oxidant stress response kinase-1 during the initial stages of chemical anoxia-induced necrotic cell death . Requirement for dual inputs of oxidant stress and increased cytosolic {Ca2+}; Pombo CM et al.; Signal transduction mechanisms activated during the early stages of necrotic cell death are poorly characterized . We have recently identified the Sterile 20 (Ste20)-like oxidant stress response kinase-1, SOK-1, which is a member of the Ste20 kinase family . We report that SOK-1 is markedly activated as early as 20 min after chemical anoxia induced by exposure of Madin-Darby canine kidney or LLC-PK1 renal tubular epithelial cells to 2-deoxyglucose (2-DG) and any one of three inhibitors of the electron transport chain, cyanide (CN), rotenone, or antimycin A . Since oxidant stress activates SOK-1, we postulated that reactive oxygen species (ROS), which are produced by the electron transport chain during chemical anoxia, might be responsible for SOK-1 activation . The time course of CN/2-DG-induced SOK-1 activation and of production of ROS, measured in cells loaded with dichlorofluorescein, were compatible with a role for ROS in SOK-1 activation . Furthermore, preincubation of LLC-PK1 cells with three unrelated scavengers of ROS, pyrrolidine dithiocarbamate, pyruvate, or nordihydroguaiaretic acid, reduced both cellular oxidant stress and activation of SOK-1 by CN/2-DG . An increase in cytosolic free {Ca2+} ({Ca2+}i) was necessary but not sufficient for CN/2-DG-induced activation of SOK-1 . Preincubation of cells with BAPTA-AM prevented activation of SOK-1 . Incubation of cells with thapsigargin or the calcium ionophore, A23187, had no effect on SOK-1 activity, but preincubation of cells with either of these agents markedly enhanced CN/2-DG-induced activation of SOK-1 (20-fold versus 7-fold) . In summary, chemical anoxia activates SOK-1 via an oxidant stress-dependent mechanism that is both critically dependent upon and markedly amplified by an increase in {Ca2+}i . This requirement for dual inputs of oxidant stress and an increase in {Ca2+}i may prevent inappropriate activation of the kinase by milder degrees of oxidant stress, which are insufficient to generate an increase in {Ca2+}i . The activation of SOK-1 may be one of the cell's earliest responses to inducers of necrotic cell death.

J Biol Chem, 1997 Nov 14, 272(46), 29200 - 6
The coatomer protein beta'-COP, a selective binding protein (RACK) for protein kinase Cepsilon; Csukai M et al.; Distinct subcellular localization of activated protein kinase C (PKC) isozymes is mediated by their binding to isozyme-specific RACKs (receptors for activated C-kinase) . Our laboratory has previously isolated one such protein, RACK1, and demonstrated that this protein displays specificity for PKCbeta . We have recently shown that at least part of the PKCepsilon RACK-binding site on PKCepsilon lies within the unique V1 region of this isozyme (Johnson, J . A., Gray, M . O., Chen, C.-H., and Mochly-Rosen, D . (1996) J . Biol . Chem . 271, 24962-24966) . Here, we have used the PKCepsilon V1 region to clone a PKCepsilon-selective RACK, which was identified as the COPI coatomer protein, beta'-COP . Similar to RACK1, beta'-COP contains seven repeats of the WD40 motif and fulfills the criteria previously established for RACKs . Activated PKCepsilon colocalizes with beta'-COP in cardiac myocytes and binds to Golgi membranes in a beta'-COP-dependent manner . A role for PKC in control of secretion has been previously suggested, but this is the first report of direct protein/protein interaction of PKCepsilon with a protein involved in vesicular trafficking.

Eur J Biochem, 1997 Oct 1, 249(1), 239 - 47
Human and murine serine-palmitoyl-CoA transferase--cloning, expression and characterization of the key enzyme in sphingolipid synthesis; Weiss B et al.; Serine palmitoyltransferase (SPT, EC 2.3.1.50) is the key enzyme in sphingolipid biosynthesis . It catalyzes the pyridoxal-5'-phosphate-dependent condensation of L-serine and palmitoyl-CoA to 3-oxosphinganine . Human expressed-sequence-tag (EST) clones are similar to the two yeast genes for synthesis of long-chain bases, LCB1 and LCB2, which are believed to encode two subunits of SPT {Buede, R., Pinto, W . J., Lester, R . L . & Dickson, R . C . (1991) J . Bacteriol . 173, 4325-5332; Nagiec, M . M., Baltisberger, J . A., Wells, G . B., Lester, R . L . & Dickson, R . C . (1994) Proc . Natl Acad . Sci . USA 91, 7899-7902} . We have cloned and characterized two complete human and murine cDNA sequences named hLCB1 & mLCB1 and hLCB2 & mLCB2, respectively, similar to the yeast LCB1 and LCB2 genes . Human embryonic kidney cells (HEK 293) transfected with murine sequences of LCB1 (mLCB1) and LCB2 (mLCB2) independently and in coexpression showed an overexpression of the transcripts on the mRNA and protein level . The enzymatic activity of cells expressing mLCB2 alone or coexpressed with mLCB1 was three times higher than the activity of untransfected HEK cells . mLCB1 expression was not required for the synthesis of 3-oxo-sphinganine in mammalian cells . Transcription/translation in vitro yielded mLCB1 (53 kDa) and mLCB2 (63 kDa) . The two proteins do not contain a signal peptide nor are they glycosylated . The endogenous and overexpressed SPT activity were both sensitive to common SPT inhibitors . Labeling studies with {1-(14)C}palmitic acid indicated that cell lines transfected with mLCB2 preferentially use the excess sphingoid bases for glucocerebroside and galactocerebroside synthesis . Our results provide conclusive genetic and biochemical evidence that the human and murine LCB2 genes described here encode serine palmitoyltransferase . Further studies will be required to unravel the function of the LCB1 gene in mammalian cells.

Eur J Biochem, 1997 Oct 1, 249(1), 142 - 8
Peptide fragments of DNA topoisomerase II with helix-forming and coiled-coil-forming properties act as inhibitors of the enzyme; Frere-Gallois V et al.; We have previously shown that a synthetic peptide (dL) consisting of amino acids 1013-1056 of human alpha topoisomerase II adopted an alpha-helix structure and formed a stable dimer coiled-coil in solution {Frere, V., Sourgen . F., Monnot, M., Troalen, F . & Fermandjian, S . (1995) J . Biol . Chem . 270, 17502-17507} . Here we studied two peptides, dP and dLshort, which are related to dL but which have a double substitution Leu1026-->Pro, Leu1037-->Pro and a deletion of the 15 C-terminal residues, respectively . The peptides were studied for their ability to form alpha-helix structures, coiled coils, and to inhibit topoisomerase II activity . In combining circular dichroism spectra with AGADIR prediction for helix structures, we demonstrated that the dLshort peptide, like its parent dL peptide, adopts an alpha-helix structure and can autoassociate into coiled-coils, while dP is completely devoid of such properties . Remarkably, only the dL and dLshort peptides act as good inhibitors of topoisomerase II in various in vitro assays . However, the dLshort peptide has a stronger helix potential and behaves as a much more potent inhibitor (5 microM versus 200 microM) compared to the dL peptide . All these data strongly suggest that the greater inhibitory effect demonstrated by the dLshort peptide is related to its higher ability to form a stable amphiphilic helix, which in turn better recognizes its homologous helical segment in topoisomerase II . Finally, we propose that the dL and the dLshort peptides could interfere with the enzymatic activity of topoisomersase II in modifying its autoassociation or translocation properties . Such peptides may serve as useful models for developing simpler and more specific inhibitors of topoisomerase II.

Eur J Biochem, 1997 Oct 1, 249(1), 85 - 91
The BH3 domain of Bax is sufficient for interaction of Bax with itself and with other family members and it is required for induction of apoptosis; Simonen M et al.; bax is an apoptosis-inducing member of the bcl-2 multigene family . We have studied interactions of human Bax with itself, and with the apoptosis-preventing members Bcl-2 and Bcl-xL using a yeast two-hybrid system . Exhaustive Bax truncations were constructed and their interactions with full-length family members studied . Bax interacted similarly with itself as with the apoptosis-suppressing family members Bcl-2 and Bcl-xL in quantitative two-hybrid studies . A region of 41 amino acids covering the recently discovered BH3 domain of Bax was found to be necessary and sufficient for all interactions of Bax . Bax truncations containing BH3, but lacking BH1 and BH2 homology domains, interacted with the other family members markedly more strongly than full-length Bax, which may reflect conformational changes required for the interactions of full-length Bax . The minimum requirements for Bax homodimerization were found to be the BH3 domain from one Bax molecule and a region covering BH3 plus BH1 from another . We also studied the apoptosis-inducing activity of the Bax truncations upon microinjection of expression plasmids into rat fibroblasts . The BH3 region was required for the apoptosis-inducing activity of Bax, whereas BH1, BH2 and the N-terminus of Bax were dispensable.

J Biol Chem, 1997 Nov 7, 272(45), 28198 - 201
Preferential interaction of sentrin with a ubiquitin-conjugating enzyme, Ubc9; Gong L et al.; Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1 . We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H . P., and Yeh, E . T . H . (1997) J . Biol . Chem . 272, 14001-14004) . Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur . To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait . A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9 . The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin . This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF . In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase . A beta-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay . Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate . Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway.

Biochemistry (Mosc), 1997 Aug, 62(8), 850 - 7
Specific cleavage of hybrid proteins by proteinase encoded by the KEX2 gene; Bessmertnaya LYa et al.; A method for isolation of the KEX2-gene-encoded membrane-bound proteinase from alpha-cells of Saccharomyces cerevisiae yeast has been modified . The isolated enzyme hydrolyzes peptides and proteins with basic amino acid pairs which are cleaved at the C-ends of their peptide bonds . Because KEX2 proteinase is located within the Golgi compartment, it may be isolated by differential centrifugation of broken cells at 7000g for 15 min and at 20,000g for 15 min . By extracting the fraction that contains the active enzyme by a detergent solution, a protein has been obtained with specific activity 30 times higher than that of the membrane extract prepared according to the standard technique . This protocol decreases the number of steps required to isolate the enzyme . The effects of pH and inhibitors on KEX2 proteinase-catalyzed hydrolysis of Ac-Leu-Lys-Arg-pNA were studied . KEX2 proteinase can participate in peptide hormone processing because it cleaves human proinsulin at the peptide bond between Arg32 and Glu33 . The KEX2 proteinase can specifically cleave large recombinant proteins, for example, a protein consisting of a gamma-interferon fragment linked to HIV1-proteinase via a Lys-Arg-containing peptide.

Biochem J, 1997 Oct 15, 327 ( Pt 2), 545 - 52
Regulation of inositol lipid-specific phospholipase cdelta by changes in Ca2+ ion concentrations; Allen V et al.; Studies of inositol lipid-specific phospholipase C (PLC) have elucidated the main regulatory pathways for PLCbeta and PLCgamma but the regulation of PLCdelta isoenzymes still remains obscure . Here we demonstrate that an increase in Ca2+ ion concentration within the physiological range (0.1-10 microM) is sufficient to stimulate PLCdelta1, but not PLCgamma1 and PLCbeta1, to hydrolyse cellular inositol lipids present in permeabilized cells . The activity of PLCdelta1 is further enhanced in the presence of phosphatidylinositol transfer protein (PI-TP) . Both full activation by Ca2+ ions and stimulation in the presence of PI-TP require an intact PH domain involved in the membrane attachment of PLCdelta1 . The physiological implication of this study is that PLCdelta1 could correspond to a previously uncharacterized PLC responsible for Ca2+ ion-stimulated inositol lipid hydrolysis observed in many cellular systems.

Spectrochim Acta A Mol Biomol Spectrosc, 1997 Sep, 53A(10), 1633 - 6
Study on the binding of cerium(III) ion and spermine to TRNA(Phe); Meng JX et al.; Fluorescence of Ce(III) aqua ion at pH 6.0 is found to be in good linear relationship with its concentration, and the intensity is strong enough to be employed to determine its concentration . TRNA(Phe) evidently quench this fluorescence . Fluorescence titration experiments were performed to Ce(III)-tRNA(Phe) system, the binding number and association constant was estimated with a Scatchard plot . Two classes of binding sites with association constant of 5.2 x 10(7) and 4.4 x 10(6) M, respectively, were found . Addition of spermine slightly decrease binding number and association constant of Ce(III) ion.

Gene, 1997 Oct 15, 199(1-2), 181 - 94
Ku80 gene expression is Sp1-dependent and sensitive to CpG methylation within a novel cis element; Ludwig DL et al.; The Ku70/80 complex, known as Ku, constitutes the DNA end binding component of the DNA-dependent protein kinase (DNA-PK) . We have characterized the promoter region of the mouse and human Ku80 genes to delineate transcriptional elements necessary for basal gene expression and proliferation-dependent regulation . Consensus Sp1 recognition elements were identified in both promoters, and were determined to be essential for basal expression . We further identified a near-perfect palindrome of 21 base pairs located immediately 5' to one Sp1 element . This sequence was present once within the mouse Ku80 promoter and seven times, in a head-to-tail tandem array, within the human Ku80 promoter . This sequence possessed homology with a methylation-sensitive promoter element, Enh2, present in the LTR of mouse intractisternal A-particles . Promoter deletion studies and expression analysis of in-vitro methylated reporter gene constructs provided strong evidence that, in vivo, this repeat sequence regulates Ku80 gene expression in cis, through a mechanism involving CpG methylation . Evidence is also presented, suggesting that Ku is directly involved in this regulatory process.

Gene, 1997 Oct 15, 199(1-2), 139 - 43
Drosophila DPP2C1, a novel member of the protein phosphatase 2C (PP2C) family; Dick T et al.; We report the molecular cloning, chromosome mapping and developmental transcription pattern of a putative serine/threonine protein phosphatase 2C (PP2C), DPP2C1, from Drosophila melanogaster . The 6-kb transcript of this first Drosophila PP2C gene encodes a 1428-aa deduced protein . The DPP2C1 protein contains a approximately 330-aa PP2C-like catalytic domain flanked by extensive N- and C-terminal sequences showing no similarities to other PP2Cs . The dpp2c1 gene maps to 4E1-2 on the X chromosome, 1.5 kb upstream of the ddlc1 gene . Northern blot analyses showed that dpp2c1 transcription is developmentally regulated, accumulating maximally during early (0-6 h) and late (12-24 h) embryogensis . The presented molecular characterisation provides the basis for a genetic dissection of DPP2C1 function.

Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12580 - 5
The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase; Yu J et al.; The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides . In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins . In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation . In this study, using a modified recombinant vaccinia transient transfection system in conjunction with a composite cDNA prepared by 5' extension of an existing GenBank sequence, we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer . hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both encoding a protein of 395 amino acids that varies in cells with their ability to couple GPIs to proteins . The gene spans approximately 25 kb of DNA on chromosome 1 . Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins . Also, hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase.

Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12521 - 6
CDC45 is required in conjunction with CDC7/DBF4 to trigger the initiation of DNA replication; Owens JC et al.; The initiation of DNA replication in Saccharomyces cerevisiae requires the protein product of the CDC45 gene . We report that although Cdc45p is present at essentially constant levels throughout the cell cycle, it completes its initiation function in late G1, after START and prior to DNA synthesis . Shortly after mitosis, cells prepare for initiation by assembling prereplicative complexes at their replication origins . These complexes are then triggered at the onset of S phase to commence DNA replication . Cells defective for CDC45 are incapable of activating the complexes to initiate DNA replication . In addition, Cdc45p and Cdc7p/Dbf4p, a kinase implicated in the G1/S phase transition, are dependent on one another for function . These data indicate that CDC45 functions in late G1 phase in concert with CDC7/DBF4 to trigger initiation at replication origins after the assembly of the prereplicative complexes.

Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12473 - 8
Cells that register logical relationships among proteins; Xu CW et al.; Two-hybrid methods have augmented the classical genetic techniques biologists use to assign function to genes . Here, we describe construction of a two-bait interaction trap that uses yeast cells to register more complex protein relationships than those detected in existing two-hybrid systems . We show that such cells can identify bridge or connecting proteins and peptide aptamers that discriminate between closely related allelic variants . The protein relationships detected by these cells are analogous to classical genetic relationships, but lend themselves to systematic application to the products of entire genomes and combinatorial libraries . We show that, by performing logical operations on the phenotypic outputs of these complex cells and existing two-hybrid cells, we can make inferences about the topology and order of protein interactions . Finally, we show that cells that register such relationships can perform logical operations on protein inputs . Thus these cells will be useful for analysis of gene and allele function, and may also define a path for construction of biological computational devices.

Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12413 - 8
Dystrobrevin and dystrophin: an interaction through coiled-coil motifs; Sadoulet-Puccio HM et al.; Dystrobrevin, a dystrophin-related and -associated protein, has been proposed to be important in the formation and maintenance of the neuromuscular junction . Dystrobrevin coprecipitates with both the acetylcholine receptor complex as well as the dystrophin glycoprotein complex . Although the nature of dystrobrevin's association with the dystrophin glycoprotein complex remains unclear, it is known that dystrobrevin binds directly to the syntrophins, a heterologous group of dystrophin-associated proteins . Using the yeast two-hybrid system to identify protein-protein interactions, we present evidence for the heterodimerization of dystrobrevin directly with dystrophin . The C terminus of dystrobrevin binds specifically to the C terminus of dystrophin . We further refined this site of interaction to these proteins' homologous coiled-coil motifs that flank their respective syntrophin-binding sites . We also show that the interaction between the dystrobrevin and dystrophin coiled-coil domains is specific and is not due to a nonspecific coiled-coil domain interaction . From the accumulated evidence of protein-protein interactions presented here and elsewhere, we propose a partially revised model of the organization of the dystrophin-associated glycoprotein complex.

Toxicol Appl Pharmacol, 1997 Nov, 147(1), 93 - 100
Additive estrogenic activities of a binary mixture of 2',4',6'-trichloro- and 2',3',4',5'-tetrachloro-4-biphenylol; Ramamoorthy K et al.; The estrogenic activity of 2',4',6'-trichloro-4-biphenylol (HO-PCB3), 2',3',4',5'-tetrachloro-4-biphenylol (HO-PCB4), and an equimolar mixture of both compounds (HO-PCB3/HO-PCB4) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 and MDA-MB-231 human breast cancer cells, HepG2 cells, and in a yeast-based reporter gene assay . Treatment of the animals with 17beta-estradiol (E2) (0.02 microg/kg/day x3) resulted in increased uterine wet weight, peroxidase activity and progesterone receptor binding . Treatment with 18, 73, 183 or 366 micromol/kg (x3) doses of HO-PCB3, HO-PCB4, or HO-PCB3/HO-PCB4 (equimolar) caused a dose-dependent increase in estrogenic activity; a maximal-induced response was not observed at any dose and the activity of the mixture was additive . Binding of E2, HO-PCB3, HO-PCB4, and HO-PCB3/HO-PCB4 to the mouse uterine estrogen receptor (ER) was determined in a competitive binding assay using {3H}E2 as the radioligand . The IC50 values were 1.1 x 10(-8), 3.4 x 10(-6), 9.9 x 10(-7), and 4.25 x 10(-6) m, respectively . HO-PCB3 and HO-PCB4 maximally induced MCF-7 cell proliferation, rat creatine kinase, and human complement C3 (C3-LUC) reporter gene activity at concentrations of 10(-5) to 10(-6) m, and these compounds were 10(3) to 10(4) less potent than E2 . The HO-PCB3/HO-PCB4 mixture was active at the high concentration (10(-5) m) and was additive for these responses . HO-PCB3 and HO-PCB4 also exhibited estrogenic activity in human HepG2 cells cotransfected with C3-LUC and an ER expression plasmid, and the estrogenic activity of the HO-PCB mixture was additive . Similar results were obtained in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene . The effects of variable ER expression on the potential synergistic interactions of HO-PCB3/HO-PCB4 were investigated in HepG2 cells cotransfected with C3-LUC (405 ng/well) and variable amounts of ER expression plasmid (270, 27, 2.7, or 0.27 ng/well) . The results show that as ER levels decreased, the magnitude of the induction response by E2, HO-PCB3, HO-PCB4, and HO-PCB3/HO-PCB4 also decreased . However, the activities of the HO-PCB mixture were additive at high and low levels of ER . Similar results were obtained in MDA-MB-231 cells cotransfected with C3-LUC and variable amounts of ER expression plasmid . The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus; MCF-7, HepG2, and MDA-MBA-231 human cancer cells; and a yeast based-reporter gene assay, both HO-PCB3 and HO-PCB4 exhibited estrogenic activity . The estrogenic activity of an equimolar mixture of these compounds was additive at high and low levels of ER expression .

Mol Plant Microbe Interact, 1997 Nov, 10(8), 984 - 93
Targeted disruption of a fungal G-protein beta subunit gene results in increased vegetative growth but reduced virulence; Kasahara S et al.; Targeted disruption of two G-protein alpha subunit genes in the chestnut blight fungus Cryphonectria parasitica revealed roles for the Gi alpha subunit CPG-1 in fungal reproduction, virulence, and vegetative growth . A second G alpha subunit, CPG-2, was found to be dispensable for these functions . We now report the cloning and targeted disruption of a C . parasitica G-protein beta subunit gene . The deduced amino acid sequence encoded by this gene, designated cpgb-1, was found to share 66.2, 65.9, and 66.7% amino acid identity with G beta homologues from human, Drosophila, and Dictyostelium origins, respectively, but only 39.7% identity with the Saccharomyces cerevisiae G beta homologue STE4 product . Low stringency Southern hybridization failed to detect any related G beta subunit genes in C . parasitica . Targeted disruption of cpgb-1 resulted in several of the changes previously reported to accompany disruption of the C . parasitica Gi alpha subunit gene cpg-1 . These included very significant reductions in pigmentation, asexual sporulation, and virulence . In contrast to results obtained for Gi alpha gene disruption, the reduction in virulence resulting from the disruption of a G beta gene was accompanied by increased, rather than decreased, vegetative growth on synthetic medium . The relevance of these results to mechanisms of fungal virulence is considered.

Microbiology, 1997 Oct, 143 ( Pt 10), 3263 - 72
Dipeptidyl aminopeptidase processing and biosynthesis of alkaline extracellular protease from Yarrowia lipolytica; Matoba S et al.; Alkaline extracellular protease (AEP) from Yarrowia lipolytica is synthesized as a precursor with a 157 aa prepro-region . Signal peptide cleavage was shown to occur after Ala15 by N-terminal amino acid radiosequencing of the largest intracellular AEP precursor . AEP proteolytic activity was not required for AEP processing . After a change of the putative active site Ser to Ala, inactive AEP with the same mobility on SDS-PAGE as wild-type mature AEP was secreted . The role of dipeptidyl aminopeptidase (DPAPase) activity in AEP processing was also investigated . Mutations early in the -X-Ala- and -X-Pro- dipeptide stretch (Pro17 to Met which should prevent DPAPase processing and Ala19 to Val which should allow removal of only the first dipeptide) did not prevent synthesis of active mature AEP nor did use of the DPAPase inhibitor ProboroPro . Deletion of the entire dipeptide stretch (Ala16 to Pro33) resulted in intracellular accumulation of an AEP precursor, which surprisingly was not glycosylated, and little or no secretion of AEP-related polypeptides . Expression of AEP in wild-type and dpp1 dap2 Saccharomyces cerevisiae strains (lacking both the Golgi and vacuolar DPAPases) resulted in secretion of only mature AEP and no AEP precursors . Transit times and levels of AEP secretion were similar for both strains . These results indicate that the KEX2-like cleavage after Lys156-Arg157, which yields mature active AEP can occur in the absence of DPAPase processing and that DPAPase processing is not necessary for secretion of mature active AEP.

J Biol Chem, 1997 Nov 7, 272(45), 28695 - 703
Cloning and characterization of a human STE20-like protein kinase with unusual cofactor requirements; Schinkmann K et al.; We cloned and characterized a novel human member of the STE20 serine/threonine protein kinase family named mst-3 . Based on its domain structure, mst-3 belongs to the SPS1 subgroup of STE20-like proteins, which includes germinal center (GC) kinase, hematopoietic progenitor kinase (HPK), kinase homologous to STE20/SPS-1 (KHS), kinases responsive to stress (KRS1/2), the mammalian STE20-like kinases (mst1/2), and the recently published STE20/oxidant stress response kinase SOK-1 . mst-3 is most closely related to SOK-1, with 88% amino acid similarity in the kinase domain . The similarity of the mst-3 kinase domain to STE20 is 42% . The mst-3 transcript is ubiquitously expressed, and the protein was found in all human, mouse, and monkey cell lines tested . An in vitro kinase assay showed that mst-3 can phosphorylate basic exogenous substrates as well as itself . Interestingly, mst-3 prefers Mn2+ to Mg2+ as a divalent cation and can use both GTP and ATP as phosphate donors . Like SOK-1, mst-3 is activated by autophosphorylation . However, a physiological stimulus of mst-3 activity was not identified . mst-3 activity does not change upon exposure to several mitogenic and stress stimuli . Overexpression of mst-3 wild-type or kinase dead protein affects neither the extracellular signal-regulated kinases (ERK1/2 or ERK6), c-Jun N-terminal kinase (JNK), p38, nor pp70S6 kinase, suggesting that mst-3 is part of a novel signaling pathway.

J Biol Chem, 1997 Nov 7, 272(45), 28501 - 11
Cell cycle-regulated expression, phosphorylation, and degradation of p55Cdc . A mammalian homolog of CDC20/Fizzy/slp1; Weinstein J; p55Cdc is a mammalian protein that shows high homology to the cell cycle proteins Cdc20p of Saccharomyces cerevisiae and the product of the Drosophila fizzy (fzy) gene, both of which contain WD repeats and are thought to be required for the metaphase-anaphase transition . The fzy mutants exhibit a metaphase arrest phenotype, which is accompanied by stabilization of cyclins A and B, leading to the hypothesis that fzy function is required for cell cycle-regulated ubiquitin-mediated proteolysis . p55Cdc expression was initiated at the G1/S transition and steady state levels of p55Cdc were highest at M and lowest in G1 . Inhibition of the 26 S proteasome prevented both mitotic exit and loss of p55Cdc at the M/G1 transition, suggesting that p55Cdc degradation was mediated by the cell cycle-regulated proteolytic pathway . Immune complexes of p55Cdc obtained at different cell cycle stages showed a variety of proteins with dramatic differences observed in the pattern of associated proteins during the transition from G2 to M . Immunolocalization of p55Cdc demonstrated dynamic changes in p55Cdc localization as the cells transit mitosis . p55Cdc appears to act as a regulatory protein interacting with several other proteins, perhaps via its seven WD repeats, at multiple points in the cell cycle.

J Biol Chem, 1997 Nov 7, 272(45), 28407 - 14
HRX leukemic fusion proteins form a heterocomplex with the leukemia-associated protein SET and protein phosphatase 2A; Adler HT et al.; One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene at chromosome locus 11q23, resulting in HRX fusion proteins . Using the yeast two-hybrid system, in vitro binding studies, and human cell culture coimmunoprecipitation experiments, we show here that a region of the HRX protein that is consistently retained in HRX leukemic fusion proteins interacts directly with SET, another protein implicated in leukemia . We have identified the binding sites on HRX for SET and show that these sequences are clustered near the A.T hooks that have been shown to bind DNA . We also show that carboxyl-terminal SET sequences, possibly the acidic tail of SET, bind to HRX . We have also found serine/threonine-specific protein phosphatase activity in anti-HRX coimmunoprecipitates . Using the phosphatase inhibitor okadaic acid and Western blotting, the phosphatase was identified as protein phosphatase 2A (PP2A) . Mutation of a single amino acid in one of the SET binding sites of HRX resulted in lower amounts of both coimmunoprecipitated SET protein and coimmunoprecipitated PP2A . These results suggest that the leukemogenic effects of HRX fusion proteins may be related to interactions with SET and PP2A.

J Biol Chem, 1997 Nov 7, 272(45), 28247 - 51
Identification of HsORC4, a member of the human origin of replication recognition complex; Quintana DG et al.; A new member of human origin recognition complex (ORC) has been cloned and identified as the human homologue of Saccharomyces cerevisiae ORC4 . HsORC4 is a 45-kDa protein encoded by a 2.2-kilobase mRNA whose amino acid sequence is 29% identical to ScORC4 . HsORC4 has a putative nucleotide triphosphate binding motif that is not seen in ScORC4 . HsORC4P also reveals an unsuspected homology to the ORC1-Cdc18 family of proteins . HsORC4 mRNA expression and protein levels remain constant through the cell cycle . HsORC4P is coimmunoprecipitated from cell extracts with another subunit of human ORC, HsORC2P, consistent with it being a part of the putative human origin recognition complex.

Curr Opin Biotechnol, 1997 Oct, 8(5), 629 - 34
Gene expression systems in the development of high-throughput screens; Jayawickreme CK et al.; Recent advances in the development of combinatorial automated chemical synthesis, robotic sample handling, and data collection and analysis have significantly increased the number of compounds available for screening against potential therapeutic targets . The implementation of highly sensitive in vitro biochemical and cell-based high-throughput screening assays is essential to facilitate the rapid identification of selective and potent lead molecules from compound libraries . The ability to easily produce functional proteins in sufficient quantities for in vitro biochemical assays and to devise useful cell-based systems is dependent on the successful application of a variety of gene expression systems.

Nature, 1997 Oct 30, 389(6654), 974 - 8
The cerebellar leucine-rich acidic nuclear protein interacts with ataxin-1; Matilla A et al.; Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells . SCA1 belongs to a growing group of neurodegenerative disorders caused by expansion of CAG repeats, which encode glutamine . Although the proteins containing these repeats are widely expressed, the neurodegeneration in SCA1 and other polyglutamine diseases selectively involves a few neuronal subtypes . The mechanism(s) underlying this neuronal specificity is unknown . Here we show that the cerebellar leucine-rich acidic nuclear protein (LANP) interacts with ataxin-1, the SCA1 gene product . LANP is expressed predominantly in Purkinje cells, the primary site of pathology in SCA1 . The interaction between LANP and ataxin-1 is significantly stronger when the number of glutamines is increased . Immunofluorescence studies demonstrate that both LANP and ataxin-1 colocalize in nuclear matrix-associated subnuclear structures . The features of the interaction between ataxin-1 and LANP, their spatial and temporal patterns of expression, and the colocalization studies indicate that cerebellar LANP is involved in the pathogenesis of SCA1.

Pharmacogenetics, 1997 Oct, 7(5), 381 - 90
Role of CYP2D6 in the N-hydroxylation of procainamide; Lessard E et al.; Sequential oxidations at the arylamine moiety of the procainamide molecule leading to the formation of N-hydroxyprocainamide and its nitroso derivative may be responsible for lupus erythematosus observed in patients treated with the drug . The objective of the present study was to characterize major cytochrome P450 isozyme(s) involved in the N-hydroxylation of procainamide . Firstly, incubations were performed with microsomes from either lymphoblastoid cells or yeast transfected with cDNA encoding for specific human cytochrome P450 isozymes . Experiments performed with these enzyme expression systems indicated that the highest formation rate of N-hydroxyprocainamide was observed in the presence of CYP2D6 enriched microsomes . Additional experiments demonstrated that the formation rate of N-hydroxyprocainamide by CYP2D6 enriched microsomes was decreased from 45 +/- 4% to 93 +/- 1% by quinidine at concentrations ranging from 30 nM to 100 microM (all p < 0.05 vs control) and by approximately 75% by antibodies directed against CYP2D6 . Secondly, incubations were performed with microsomes prepared from 15 human liver samples . Using this approach, an excellent correlation was observed between the formation rate of N-hydroxyprocainamide and dextromethorphan O-demethylase activity (CYP2D6; r = 0.9305; p < 0.0001) . In contrast, no correlation could be established between N-hydroxyprocainamide formation rate and caffeine N3-demethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), S-mephenytoin N-demethylase (CYP2B6), tolbutamide methlhydroxylase (CYP2C9), S-mephenytoin 4'-hydroxylase (CYP2C19), chlorzoxazone 6-hydroxylase (CYP2E1), dextromethorphan N-demethylase (CYP3A4), testosterone 6 beta-hydroxylase (CYP3A4/5) or lauric acid 12-hydroxylase (CYP4A11) activities . Furthermore, formation rate of N-hydroxyprocainamide was decreased in a concentration-dependent manner by quinidine (300 nM to 100 microM) and by antibodies directed against CYP2D6 but not by furafylline 20 microM (CYP1A2), ketoconazole 1 microM (CYP3A4), sulfaphenazole 10 microM (CYP2C9) or antibodies directed against CYP1A1/1A2, CYP2C, CYP2A6, CYP2E1 or CYP3A4/3A5 . In conclusion, the results obtained in the present study demonstrate that CYP2D6 is the major human cytochrome P450 isozyme involved in the formation of the reactive metabolite of procainamide, namely N-hydroxyprocainamide.

Mech Ageing Dev, 1997 Dec, 98(3), 223 - 30
Molecular genetic approaches to the genes of longevity, aging and neurodegeneration in mammals; Mori N; Accumulative evidence suggests that species life-span is determined, at least in part, genetically . Recent cloning works using yeast (Saccharomyces cerevisiae) and worms (Caenorhabditis elegans) revealed several potential candidate genes, e.g . SIR4, a transcriptional silencing factor, and age-1, a putative signal transduction molecule, respectively, that may be involved in determining and/or regulating species life-span in lower organisms . It is, however, not clear yet whether mammalian homologs of these genes are also relevant to controlling longevity in higher organisms . In mice and humans several silencing factors are essential for cell-type specific gene expression . A variety of signal transducing molecules are also known to play important roles in mammals . I will briefly summarize recent progress in molecular genetic studies on such longevity-related genes, and discuss these results with our recent findings on a neural-selective silencing factor and a neural-specific signaling molecule that are important for functioning of the nervous system.

Int Immunol, 1997 Oct, 9(10), 1607 - 13
Molecular genetic characterization of XRCC4 function; Mizuta R et al.; XRCC4 is a generally expressed protein of 334 amino acids that is involved in the repair of DNA double-strand breaks and in V(D)J recombination, but its function is unknown . In this study, we have used a mutational approach and the yeast two-hybrid method to perform an initial characterization of this protein . We show that the XRCC4 protein is located in the nucleus . We also demonstrate that several potential phosphorylation sites are not required for XRCC4 function in a transient V(D)J recombination assay . In addition, we show that XRCC4 forms a homodimer in vivo with the homodimerization domain being located within amino acids 115-204 . Finally, we define a core domain of XRCC4 that functions in V(D)J recombination and comprises amino acids 18-204 . Potential functions of XRCC4 are discussed.

Curr Top Dev Biol, 1998, 37, 201 - 39
Caught in the act: deducing meiotic function from protein immunolocalization; Ashley T et al.; Meiotic division comprises a complex series of events, many of which are unique in the life cycle of the organism . The process utilizes both proteins that participate in normal mitotic cell cycle progression and DNA damage repair and proteins expressed only during meiosis . Until recently, few meiotic protein participants had been identified and characterized, but several recent developments have changed this situation . Proteins can be selected for study based on their cDNA sequence and similarity to known proteins with "suspicious" repair/recombination or cell cycle activity and antibodies against these proteins applied to meiotic nuclei to test for activity . With the development of gene sequence data bases from many organisms, similarity to a known protein need not be based on the same or even a closely related species . Potential interactions between two or more proteins can be identified and involvement in a common process inferred based on antibody colocalization . The gene sequence can be disrupted and the effect on meiotic progression directly examined . Previously identified structures, the synaptonemal complex (SC) and both early and late recombination nodules (RNs), provide structural and temporal landmarks that assist in inferring meiotic activity of the protein being studied . Mammalian meiosis is especially attractive for these kinds of studies since spermatocyte and oocyte nuclei are large with distinct nuclear organelles and since meiosis is highly protracted, occurring over a period of several days . In this chapter, an approach to the study of mammalian meiosis based on use of specific antibodies is outlined and methods of coupling this approach to other techniques, such as targeted gene disruption or chromosome aberrations, are described . Some of the proteins already identified as participants in meiotic prophase are reviewed and their presumed functions discussed.

Curr Top Dev Biol, 1998, 37, 117 - 40
Functions of DNA repair genes during meiosis; Cummings WJ et al.; One of the most basic functions in any organism is DNA repair . In addition, programmed DNA "damage," in the form of DNA double-strand breaks (DSBs), is a regular part of the physiology of most organisms . There are three main types of DSB repair: homologous recombination; single-strand annealing; and nonhomologous end joining . The gene products known to be required for these repair processes are conserved in evolution, but the relative dependence on different pathways for DSB repair is different when systems are compared . In the yeast Saccharomyces cerevisiae, the formation and repair of DNA double-strand breaks (DSBs) is apparently an essential feature of meiotic recombination . However, it is not clear whether DSBs are a conserved feature of meiotic recombination in eukaryotes . The basidiomycete Coprinus cinereus presents an experimental system which is amenable to genetic analysis, processes DSBs in a manner similar to complex eukaryotes, and has a naturally synchronous meiosis . An understanding of the functions of conserved genes in DSB repair in C . cinereus and other similar systems will help to determine whether DSB repair is a unifying theme in meiotic recombination or whether conserved gene products have other essential functions that tie together DNA repair and meiosis.

EMBO J, 1997 Nov 3, 16(21), 6521 - 34
Dual role for fimbriata in regulating floral homeotic genes and cell division in Antirrhinum; Ingram GC et al.; The fimbriata (fim) gene of Antirrhinum affects both the identity and arrangement of organs within the flower, and encodes a protein with an F-box motif . We show that FIM associates with a family of proteins, termed FAPs (FIM-associated proteins), that are closely related to human and yeast Skp1 proteins . These proteins form complexes with F-box-containing partners to promote protein degradation and cell cycle progression . The fap genes are expressed in inflorescence and floral meristems in a pattern that incorporates the domain of fim expression, supporting an in vivo role for a FIM-FAP complex . Analysis of a series of novel fim alleles shows that fim plays a key role in the activation of organ identity genes . In addition, fim acts in the regions between floral organs to specify the correct positioning and maintenance of morphological boundaries . Taking these results together, we propose that FIM-FAP complexes affect both gene expression and cell division, perhaps by promoting selective degradation of regulatory proteins . This may provide a mechanism by which morphological boundaries can be aligned with domains of gene expression during floral development.

EMBO J, 1997 Nov 3, 16(21), 6374 - 83
Identification of a species-specific inhibitor of glycosylphosphatidylinositol synthesis; Sutterlin C et al.; Glycosylphosphatidylinositol (GPI)-anchoring represents a mechanism for attaching proteins to the cell surface that is used among all eukaryotes . A common core structure, EthN-P-Man3-GlcN-PI, is synthesized by sequential transfer of sugars and ethanolamine-P to PI and is highly conserved between organisms . We have screened for natural compounds that inhibit GPI-anchoring in yeast and have identified a terpenoid lactone, YW3548, that specifically blocks the addition of the third mannose to the intermediate structure Man2-GlcN-acyIPI . Consistent with the block in GPI synthesis, YW3548 prevents the incorporation of {3H}myo-inositol into proteins, transport of GPI-anchored proteins to the Golgi and is toxic . The compound inhibits the same step of GPI synthesis in mammalian cells, but has no significant activity in protozoa . These results suggest that despite the conserved core structure, the GPI biosynthetic machinery may be different enough between mammalian and protozoa to represent a target for anti-protozoan chemotherapy.

EMBO J, 1997 Nov 3, 16(21), 6346 - 54
The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis; Rheaume E et al.; Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli . Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes . Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved . We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments . Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO . In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein . Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A . We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates . Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication . These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.

Br J Pharmacol, 1997 Oct, 122(3), 493 - 503
Role of peroxynitrite and activation of poly (ADP-ribose) synthase in the vascular failure induced by zymosan-activated plasma; Cuzzocrea S et al.; 1 . Zymosan is a wall component of the yeast Saccharomyces Cerevisiae . Injection of zymosan into experimental animals is known to produce an intense inflammatory response . Recent studies demonstrated that the zymosan-induced inflammatory response in vivo can be ameliorated by inhibitors of nitric oxide (NO) biosynthesis . The cytotoxic effects of NO are, in part, mediated by the oxidant preoxynitrite and subsequent activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) . In the present in vitro study, we have investigated the cellular mechanisms of vascular failure elicited by zymosan-activated plasma and the contribution of peroxynitrite production and activation of PARS to the changes . 2 . Incubation of rat aortic smooth muscle cells with zymosan-activated plasma (ZAP) induced the production of nitrite, the breakdown product of NO, due to the expression of the inducible isoform of NO synthase (iNOS) over 6 24 h . In addition, ZAP triggered the production of peroxynitrite in these cells, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123 and by nitrotyrosine Western blotting . 3 . Incubation of the smooth muscle cells with ZAP induced DNA single strand breakage and PARS activation . These effects were reduced by inhibition of NOS with NG-methyl-L-arginine (L-NMA, 3 mM), and by glutathione (3 mM), a scavenger of peroxynitrite . The PARS inhibitor 3-aminobenzamide (1 mM) inhibited the ZAP-induced activation of PARS . 4 . Incubation of thoracic aortae with ZAP in vitro caused a reduction of the contractions of the blood vessels to noradrenaline (vascular hyporeactivity) and elicited a reduced responsiveness to the endothelium-dependent vasodilator acetylcholine (endothelial dysfunction) . 5 . Preincubation of the thoracic aortae with L-NMA (1 mM), glutathione (3 mM) or by the PARS inhibitor 3-aminobenzamide (1 mM) prevented the development of vascular hyporeactivity in response to ZAP . Moreover, glutathione and 3-aminobenzamide treatment protected against the ZAP-induced development of endothelial dysfunction . The PARS-related loss of the vascular contractility was evident at 30 min after incubation in endothelium-intact, but not in endothelium-denuded vessels and also manifested at 6 h after incubation with ZAP in endothelium-denuded rings . The acute response is probably related, therefore, to peroxynitrite formation (involving the endothelial NO synthase), whereas the delayed response may be related to the expression of iNOS in the smooth muscle . 6 . The data obtained suggest that zymosan-activated plasma causes vascular dysfunction by inducing the simultaneous formation of superoxide and NO . These radicals combine to form peroxynitrite, which, in turn causes DNA injury and PARS activation . The protective effect of 3-aminobenzamide demonstrates that PARS activation contributes both to the development of vascular hyporeactivity and endothelial dysfunction during the vascular failure induced by ZAP.

J Biol Chem, 1997 Oct 31, 272(44), 27582 - 8
FKBP12 binds the inositol 1,4,5-trisphosphate receptor at leucine-proline (1400-1401) and anchors calcineurin to this FK506-like domain; Cameron AM et al.; The immunophilin FKBP12 is one of the most abundant and conserved proteins in biology . It is the primary receptor for the immunosuppressant actions of the drug FK506 in whose presence FKBP12 binds to and inhibits calcineurin, disrupting interleukin formation in lymphocytes . The physiologic functions of FKBP12 are less clear, although the protein has been demonstrated to physiologically interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the ryanodine receptor, and the type 1 transforming growth factor beta receptor . We now report that FKBP12 binds the IP3R at residues 1400-1401, a leucyl-prolyl dipeptide epitope that structurally resembles FK506 . We further demonstrate that binding to IP3R at this site enables FKBP12 to interact with calcineurin, presumably to anchor the phosphatase to IP3R and modulate the receptor's phosphorylation status . We propose that FK506 promotes an FKBP12-calcineurin interaction by mimicking structurally similar dipeptide epitopes present within proteins that use FKBP12 to anchor calcineurin to the appropriate physiologic substrates.

Biochemistry, 1997 Oct 14, 36(41), 12554 - 9
Structural and functional analysis of peptidyl oligosaccharyl transferase inhibitors; Kellenberger C et al.; The peptide cyclo(hex-Amb(1)-Cys(2))-Thr(3)-Val(4)-Thr(5)-Nph(6)-NH2 was previously shown to be a slow, tight-binding inhibitor (Ki = 37 nM) of the yeast oligosaccharyl transferase (OT) {Hendrickson et al . (1996) J . Am . Chem . Soc . 118, 7636-7637} . This enzyme catalyzes the transfer of a carbohydrate moiety to an asparagine residue in the consensus sequence Asn-Xaa-Thr/Ser . Herein we present a study of the contribution of the residues in positions 1, 3, 4, and 5 to OT binding . Replacement of the threonine (residue 3) by valine or (S)-2-aminobutyric acid dramatically reduced the potency of the inhibitor while, surprisingly, the incorporation of an additional methylene into the side chain of residue 1 {(S)-2,3-diaminobutyric acid changed to ornithine} had very little effect . Variants with acidic, basic, hydrophilic/polar, and hydrophobic side chains in positions 4 and 5 were also evaluated for both yeast and porcine liver OT inhibition . This aspect of the study reveals that basic (lysine) and acidic (glutamic acid) residues are detrimental to the binding, whereas hydrophobic (valine) and polar/hydrophilic (threonine) residues are both well tolerated . The kinetic behavior of substrate analogs {cyclo(hex-Asn(1)-Cys(2))-Thr(3)-Xaa(4)-Yaa(5)-Nph-NH2} corresponding to inhibitors of weak, medium, and strong potency was also examined in order to provide insight into the nature of these inhibitors.

J Biol Chem, 1997 Oct 31, 272(44), 28102 - 6
The Mr 18,000 subunit of the peripheral-type benzodiazepine receptor exhibits both benzodiazepine and isoquinoline carboxamide binding sites in the absence of the voltage-dependent anion channel or of the adenine nucleotide carrier; Joseph-Liauzun E et al.; The peripheral type benzodiazepine receptor (PBR) binds benzodiazepines such as RO5-4864 and isoquinoline carboxamide derivatives such as PK11195 . This receptor includes an Mr 18,000 isoquinoline-binding subunit predominantly located in mitochondrial mem- branes . This protein has been found to copurify with two other mitochondrial proteins, namely the outer membrane voltage-dependent anion channel (VDAC), also known as mitochondrial porin, and the inner membrane adenine nucleotide carrier . In vitro reconstitution experiments suggested that the PBR was a multimeric complex in which the isoquinoline binding site was on the Mr 18,000 subunit, denoted pk18, whereas the benzodiazepine binding site required the association of this subunit with VDAC to be expressed . Untransformed cells of the yeast Saccharomyces cerevisiae are devoid of specific binding sites for isoquinolines and benzodiazepines, whereas yeast cells transformed with a pk18-expressing vector exhibit RO5-4864 and PK11195 binding sites that are pharmacologically identical to those of the PBR . To clarify the role of VDAC and of the adenine nucleotide carrier, if any, in the constitution of the benzodiazepine binding site, yeast host strains were constructed in which the corresponding genes had been knocked out . Mitochondria prepared from pk18-producing cells devoid of either VDAC or adenine nucleotide carrier exhibit both benzodiazepine and isoquinoline carboxamide binding sites with little or no change in the Kd values as compared with the wild-type background . These results rule out the contention that VDAC is indispensable for establishing the benzodiazepine binding site and are in agreement with the hypothesis that the Mr 18,000 subunit carries both the isoquinoline carboxamide and benzodiazepine binding domains.

Cell, 1997 Oct 17, 91(2), 209 - 19
F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex; Skowyra D et al.; We have reconstituted the ubiquitination pathway for the Cdk inhibitor Sic1 using recombinant proteins . Skp1, Cdc53, and the F-box protein Cdc4 form a complex, SCFCdc4, which functions as a Sic1 ubiquitin-ligase (E3) in combination with the ubiquitin conjugating enzyme (E2) Cdc34 and E1 . Cdc4 assembled with Skp1 functions as the receptor that selectively binds phosphorylated Sic1 . Grr1, an F-box protein involved in Cln destruction, forms complexes with Skp1 and Cdc53 and binds phosphorylated Cln1 and Cln2, but not Sic1 . Because the constituents of the SCF complex are members of protein families, SCFCdc4 is likely to serve as the prototype for a large class of E3s formed by combinatorial interactions of related family members . SCF complexes couple protein kinase signaling pathways to the control of protein abundance.

Virology, 1997 Oct 13, 237(1), 33 - 45
HsN3 proteasomal subunit as a target for human immunodeficiency virus type 1 Nef protein; Rossi F et al.; HIV-1 Nef protein is important for pathogenicity, but its biochemical function remains obscure . To clarify its role, a yeast two-hybrid system (ths) screening was utilized to identify Nef cellular partners . Of 79 yeast clones harboring cDNAs for putative Nef binding proteins, 27 (34%) contained the coding region for HsN3 proteasomal subunit . HsN3 behaved as bona fide Nef partner in ths control crosses . Nef-HsN3 interaction was confirmed by in vitro binding experiments . In particular, recombinant Nef was able to capture the HsN3 subunit from a natural proteasome preparation . In Nef, the interacting region was mapped within aa 34-143, which span the structured portion of the protein, including the SH3-binding domain . In HsN3, Nef-binding portion was restricted to aa 73-249, and the tract 219-249-reminiscent of SH3 domain N-terminal 3/5ths-was shown to be essential, though not sufficient . Attempts to purify a Nef-HsN3 complex from transfected COS7 cells were unsuccessful . However, Nef was found to markedly downregulate intracellular levels of both a coexpressed HsN3 and the endogenous simian homologue . These results suggest that Nef, by binding to a subunit, might alter proteasome function in infected cells .

Biochem Biophys Res Commun, 1997 Oct 20, 239(2), 439 - 46
Cloning and characterization of novel CIS family genes; Masuhara M et al.; We have reported two JAK-signaling modulators, CIS (cytokine-inducible SH2 protein) and JAB (JAK2 binding protein), which are structurally related . Here we cloned three additional CIS family genes (CIS2, CIS3, and CIS4) on the basis of an expression sequence tag (EST) database search . We also found at least two additional candidates of this gene family in the database . These genes were induced by erythropoietin and granulocyte-macrophage colony stimulating factor in certain hematopoietic cell lines . The SH2 domain and a C-terminal 40 amino acid region, designated the CIS homology domain (CH domain), are highly conserved in this family, while the N-terminal regions of these proteins share little similarity . A yeast two-hybrid assay and in vitro and in vivo binding assays revealed that in addition to JAB, CIS3 bound to the JAK2 tyrosine kinase domain (JH1), although the interaction of CIS3 with the JAK2-JH1 domain was much weaker than that of JAB . Transient expression of JAB and CIS3, but not other CISs, strongly inhibited leukemia inhibitory factor (LIF)-induced STAT3-reporter gene activation in 293 cells . Furthermore, constitutive overexpression of JAB and CIS3 in M1 leukemia cells prevented LIF-induced differentiation and growth arrest . Although the physiological function remains to be investigated, CIS family genes could play a role in the negative regulation of cytokine signaling by interacting with specific targets .

Hum Mol Genet, 1997 Nov, 6(12), 2011 - 9
Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types; Kilian A et al.; Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis . Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells . We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species . This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215 . The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source . We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines . Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain . Sequencing of these products suggests that they arise by alternative splicing . Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns . Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

Carcinogenesis, 1997 Sep, 18(9), 1793 - 8
Metabolism of 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) by human cytochrome P4501B1; Crofts FG et al.; Cytochrome P4501B1 (CYP1B1) is the most recently identified member of the dioxin-inducible CYP1 family . CYP1B1 is constitutively expressed in most human tissues, including colon and breast, and can activate numerous chemically diverse carcinogens . We evaluated the metabolism of the dietary heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) by microsomes from yeast expressing the human CYP1B1 protein . PhIP metabolites were analysed by HPLC with fluorescence and absorbance detection . We found that human CYP1B1 metabolizes PhIP to three products: N2-OH-PhIP, a mutagenic activation product; 4'-OH-PhIP, a detoxification product; and 2-OH-PhIP, the mutagenic potential of which is unknown . Metabolite identity was confirmed by co-elution with authentic standards and synchronous fluorescence spectroscopy . The identity of the 2-OH-PhIP standard was additionally confirmed by mass spectrometry . Kinetic studies of the formation of N2-OH-PhIP, 4'-OH-PhIP and 2-OH-PhIP by CYP1B1 indicated apparent Km values of 5.7 +/- 1.3, 2.2 +/- 0.5 and 1.3 +/- 0.2 microM, respectively . Apparent turnover rates were 0.40 +/- 0.03, 0.93 +/- 0.02 and 0.04 +/- 0.00 nmol product/min nmol P450, respectively . At saturating levels of substrate, CYP1B1-mediated formation of the non-mutagenic metabolite 4'-OH-PhIP was favored two-fold over that of the mutagenic metabolite, N2-OH-PhIP and >10-fold over that of 2-OH-PhIP . The formation of N2-OH-PhIP, a potent mutagen implicated in the etiology of human colon and breast cancer, indicates that CYP1B1 may play an important role in PhIP-mediated carcinogenesis.

Nat Genet, 1997 Oct, 17(2), 215 - 7
Aconitase and mitochondrial iron-sulphur protein deficiency in Friedreich ataxia; Rotig A et al.; Friedreich ataxia (FRDA) is a common autosomal recessive degenerative disease (1/50,000 live births) characterized by a progressive-gait and limb ataxia with lack of tendon reflexes in the legs, dysarthria and pyramidal weakness of the inferior limbs . Hypertrophic cardiomyopathy is observed in most FRDA patients . The gene associated with the disease has been mapped to chromosome 9q13 (ref . 3) and encodes a 210-amino-acid protein, frataxin . FRDA is caused primarily by a GAA repeat expansion within the first intron of the frataxin gene, which accounts for 98% of mutant alleles . The function of the protein is unknown, but an increased iron content has been reported in hearts of FRDA patients and in mitochondria of yeast strains carrying a deleted frataxin gene counterpart (YFH1), suggesting that frataxin plays a major role in regulating mitochondrial iron transport . Here, we report a deficient activity of the iron-sulphur (Fe-S) cluster-containing subunits of mitochondrial respiratory complexes I, II and III in the endomyocardial biopsy of two unrelated FRDA patients . Aconitase, an iron-sulphur protein involved in iron homeostasis, was found to be deficient as well . Moreover, disruption of the YFH1 gene resulted in multiple Fe-S-dependent enzyme deficiencies in yeast . The deficiency of Fe-S-dependent enzyme activities in both FRDA patients and yeast should be related to mitochondrial iron accumulation, especially as Fe-S proteins are remarkably sensitive to free radicals . Mutated frataxin triggers aconitase and mitochondrial Fe-S respiratory enzyme deficiency in FRDA, which should therefore be regarded as a mitochondrial disorder.

RNA, 1997 Oct, 3(10), 1153 - 8
In vivo HIV-1 frameshifting efficiency is directly related to the stability of the stem-loop stimulatory signal; Bidou L et al.; In many retroviruses, the expression of reverse transcriptase, protease, and integrase is dependent upon a -1 frameshift event . The frameshift signal is composed of a slippery sequence where the ribosome shifts, and a downstream stimulatory sequence . In most cases, the stimulatory sequence is a pseudoknot, but in some viruses, such as human immunodeficiency virus type 1 (HIV-1), a single stem-loop is involved . Here, we analyzed the precise role of the stem-loop thermodynamic stability . We tested the frameshifting stimulatory activity of a series of HIV-1-derived sequences showing a stepwise increment of the estimated deltaG degrees . These sequences were introduced at the junction of a lacZ-luc fusion gene cloned on a versatile expression vector, and the different constructs were tested in Saccharomyces cerevisiae and in mouse NIH3T3 cells . The results showed that the frameshifting efficiency was correlated directly to the stem stability between deltaG degrees = -2.5 kcal mol(-1) and deltaG degrees = -19.4 kcal mol(-1) . This demonstrates the essential role of the stability of the stem-loop and does not support the involvement of a specific RNA-binding protein target sequence . However, increasing further the stem stability led to a diminution of frameshifting efficiency, suggesting that the stem-loop acts through a precise kinetic of pausing . Because the same pattern was observed in both yeast and mouse cells, it is likely that the stimulatory mechanism is conserved through evolution.

J Biol Chem, 1997 Oct 10, 272(41), 26009 - 16
Heat shock transcription factor 1 binds selectively in vitro to Ku protein and the catalytic subunit of the DNA-dependent protein kinase; Huang J et al.; Heat shock transcription factor 1 (HSF1) functions as the master regulator of the heat shock response in eukaryotes . We have previously shown that, in addition to its role as a transcription factor, HSF1 stimulates the activity of the DNA-dependent protein kinase (DNA-PK) . DNA-PK is composed of two components: a 460-kDa catalytic subunit and a 70- and 86-kDa heterodimeric regulatory component, also known as the Ku protein . We report here that HSF1 binds specifically to each of the two components of DNA-PK . Binding occurs in the absence of DNA . The complex with the Ku protein is stable and forms at a stoichiometry close to unity between the Ku protein heterodimer and the active HSF1 trimer . The binding is blocked by antibodies against HSF1 . Our results show that HSF1 also binds directly, but more weakly, to the catalytic subunit of DNA-PK . Both interactions are dependent on a specific region within the HSF1 regulatory domain . This sequence is necessary but not sufficient for HSF1 stimulation of DNA-PK activity . The ability of HSF1 to interact with both components of DNA-PK provides a potential mechanism for the activation of DNA-PK in response to heat and other forms of stress.

J Biol Chem, 1997 Oct 10, 272(41), 25500 - 6
Isolation and characterization of PBP, a protein that interacts with peroxisome proliferator-activated receptor; Zhu Y et al.; In an attempt to identify cofactors that could possibly influence the transcriptional activity of peroxisome proliferator-activated receptors (PPARs), we used a yeast two-hybrid system with Gal4-PPARgamma as bait to screen a mouse liver cDNA library and have identified steroid receptor coactivator-1 (SRC-1) as a PPAR transcriptional coactivator . We now report the isolation of a cDNA encoding a 165-kDa PPARgamma-binding protein, designated PBP which also serves as a coactivator . PBP also binds to PPARalpha, RARalpha, RXR, and TRbeta1, and this binding is increased in the presence of specific ligands . Deletion of the last 12 amino acids from the carboxyl terminus of PPARgamma results in the abolition of interaction between PBP and PPARgamma . PBP modestly increased the transcriptional activity of PPARgamma, and a truncated form of PBP (amino acids 487-735) acted as a dominant-negative repressor, suggesting that PBP is a genuine coactivator for PPAR . In addition, PBP contains two LXXLL signature motifs considered necessary and sufficient for the binding of several coactivators to nuclear receptors . In situ hybridization and Northern analysis showed that PBP is expressed in many tissues of adult mice, including the germinal epithelium of testis, where it appeared most abundant, and during ontogeny, suggesting a possible role for this cofactor in cellular proliferation and differentiation.

J Biol Chem, 1997 Oct 10, 272(41), 25455 - 61
Purification and characterization of the alpha-1,3-mannosylmannose-recognizing lectin of Crocus vernus bulbs; Misaki A et al.; A unique mannose-binding lectin, highly specific for terminal Man(alpha1,3)Man groups, was isolated from bulbs of crocus (Crocus vernus All.) . The lectin failed to bind to a mannose affinity column and was purified by simple gel permeation chromatography (Sephacryl S200) . The purified lectin, obtained in crystalline form, had a molecular mass of 44 kDa on gel filtration and showed a single peptide band with a molecular mass of 11 kDa on SDS-polyacrylamide gel electrophoresis, indicating it to be a tetrameric protein composed of four identical subunits . The N-terminal amino acid sequence analysis of the crocus lectin showed essentially no homology with that of other mannose-binding bulb lectins . The crocus lectin selectively interacted with the wild type Saccharomyces cerevisiae and other mannans carrying terminal Man(alpha1,3)Man but not with those lacking this disaccharide unit . In hapten inhibition studies, methyl alpha-mannopyranoside did not inhibit the mannan-lectin interaction . Of various alpha-mannooligosaccharides, those having the Man(alpha1,3)Man sequence showed the highest inhibitory potency, confirming the strict requirement of lectin for terminal alpha1,3-linked mannosylmannose units . An affinity column of immobilized lectin enabled the complete resolution of yeast mannan and glycogen . The immobilized lectin may provide a useful tool for purification and analysis of biologically important polysaccharides and glycoproteins.

J Biol Chem, 1997 Oct 10, 272(41), 25413 - 6
COPII subunit interactions in the assembly of the vesicle coat; Shaywitz DA et al.; In vitro analysis of COPII vesicle formation in the yeast Saccharomyces cerevisiae has demonstrated the requirement for three cytosolic factors: Sec31p-Sec13p, Sec23p-Sec24p, and Sar1p . Convergent evidence suggests that the peripheral endoplasmic reticulum (ER) membrane protein Sec16p also represents an important component of the vesicle assembly apparatus: SEC16 interacts genetically with all five COPII genes; Sec16p binds to Sec23p and Sec24p, is found on ER-derived transport vesicles, and is required in vitro for the efficient release of ER-derived vesicle cargo . In this report, we demonstrate an important functional interaction between Sec16p and Sec31p . First, we map onto Sec31p binding regions for Sec16p, Sec23p, Sec24p, and Sec13p . Second, we show that a truncation mutant of Sec31p specifically defective for Sec16p binding is unable to complement a sec31Delta mutant and cannot rescue the secretion defect of a temperature-sensitive sec31 mutant at nonpermissive temperatures . We propose that Sec16p organizes the assembly of a coat that is stabilized both by the interaction of Sec31p with Sec23p and Sec24p and by the interaction of these three components with Sec16p.

Plant Physiol, 1997 Sep, 115(1), 283 - 9
Rapid and transient induction of a parsley microsomal delta 12 fatty acid desaturase mRNA by fungal elicitor; Kirsch C et al.; Treatment of cultured parsley (Petroselinum crispum L.) cells with a structurally defined peptide elicitor (Pep25) of fungal origin has previously been shown to cause rapid and large changes in the levels of various desaturated fatty acids . We isolated two distinct parsley cDNAs sharing high sequence similarity with microsomal omega-6 fatty acid desaturases (FADs) . One of them was functionally identified as a delta 12 FAD by expression in the yeast Saccharomyces cerevisiae . Two dienoic fatty acids, hexadecadienoic and linoleic, which were not detectable in control cells, together constituted up to 12% of the total fatty acids in the transformed yeast cells . delta 12 FAD mRNA accumulated rapidly and transiently in elicitor-treated parsley cells, protoplasts, and leaves . These and previous results indicate that fatty acid desaturation is an important early component of the complex defense response of parsley to attempted fungal infection.

Radiat Oncol Investig, 1997, 5(4), 163 - 9
Atomic force microscope imaging of DNA and DNA repair proteins: applications in radiobiological research; Pang D et al.; By using the atomic force microscope (AFM), three-dimensional structures of biological specimens may be imaged at nanometer resolution . Furthermore, samples can be imaged in air or in fluid environments . The tapping mode of AFM operation for imaging has offered a significant advance in visualizing soft biological structures, such as DNA, proteins, and membranes . Here, we review the principles underlying the application of this instrument to radiation biological investigations . We focus on examples of proteins involved in the processes of repair of damaged DNA, including poly(ADP-ribose) polymerase, Ku protein, and DNA protein kinase . Novel observations on the character of DNA damage and repair have been addressed by direct visualization of DNA and protein-DNA interactions, such as the observation that the Ku protein is capable of physically joining DNA fragments in vitro . The AFM offers a powerful tool for investigating biologically important molecular interactions that are relevant to DNA damage and repair processes.

J Mol Biol, 1997 Oct 3, 272(4), 536 - 40
Two conformations of RNA polymerase II revealed by electron crystallography; Asturias FJ et al.; A new two-dimensional crystal form of yeast RNA polymerase II was obtained in which the conformation of the enzyme appears "open", allowing entry of DNA, as required for the initiation of transcription . By contrast, a previous crystal form contained the enzyme in a "closed" conformation, appropriate for retention of DNA during RNA chain elongation . Interaction with two polymerase subunits, Rpb4 and Rpb7, favors the closed conformation, and binding of general transcription factor TFIIE may do so as well . The effect of Rpb4 and Rpb7, together with previous biochemical evidence, leads to the conclusion that the open to closed transition is a crucial step in the transcription initiation process .

Mol Cell Biol, 1997 Nov, 17(11), 6683 - 92
A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions; Peterson AJ et al.; The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors . PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies . The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini . Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph . Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions . These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants . Analysis of site-directed point mutations identifies residues that are important for SPM domain function . These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators . In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo . We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

Mol Cell Biol, 1997 Nov, 17(11), 6574 - 84
The intermembrane space domain of mitochondrial Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences; Moczko M et al.; Mitochondrial protein import is thought to involve the sequential interaction of preproteins with binding sites on cis and trans sides of the membranes . For translocation across the outer membrane, preproteins first interact with the cytosolic domains of import receptors (cis) and then are translocated through a general import pore, in a process proposed to involve binding to a trans site on the intermembrane space (IMS) side . Controversial results have been reported for the role of the IMS domain of the essential outer membrane protein Tom22 in formation of the trans site . We show with different mutant mitochondria that a lack of the IMS domain only moderately reduces the direct import of preproteins with N-terminal targeting sequences . The dependence of import on the IMS domain of Tom22 is significantly enhanced by removing the cytosolic domains of import receptors or by performing import in two steps, i.e., accumulation of a preprotein at the outer membrane in the absence of a membrane potential (delta psi) and subsequent import after reestablishment of a delta psi . After the removal of cytosolic receptor domains, two-step import of a cleavable preprotein strictly requires the IMS domain . In contrast, preproteins with internal targeting information do not depend on the IMS domain of Tom22 . We conclude that the negatively charged IMS domain of Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences, in agreement with the acid chain hypothesis of mitochondrial protein import.

Mol Cell Biol, 1997 Nov, 17(11), 6386 - 93
Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells; Taghian DG et al.; Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting . Transcription also stimulates spontaneous recombination by an unknown mechanism . We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA . One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo . The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms) . This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels . Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1% . Strikingly, 97% of these products arose by gene conversion . Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal . DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing) . In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over . For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription . Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.

Mol Cell Biol, 1997 Nov, 17(11), 6283 - 93
HOY1, a homeo gene required for hyphal formation in Yarrowia lipolytica; Torres-Guzman JC et al.; The dimorphic fungus Yarrowia lipolytica grows to form hyphae either in rich media or in media with GlcNAc as a carbon source . A visual screening, called FIL (filamentation minus), for Y . lipolytica yeast growth mutants has been developed . The FIL screen was used to identify three Y . lipolytica genes that abolish hypha formation in all media assayed . Y . lipolytica HOY1, a gene whose deletion prevents the yeast-hypha transition both in liquid and solid media, was characterized . HOY1 is predicted to encode a 509-amino-acid protein with a homeodomain homologous to that found in the chicken Hox4.8 gene . Analysis of the protein predicts a nuclear location . These observations suggest that Hoy1p may function as a transcriptional regulatory protein . In disrupted strains, reintroduction of HOY1 restored the capacity for hypha formation . Northern blot hybridization revealed the HOY1 transcript to be approximately 1.6 kb . Expression of this gene was detected when Y . lipolytica grew as a budding yeast, but an increase in its expression was observed by 1 h after cells had been induced to form hyphae . The possible functions of HOY1 in hyphal growth and the uses of the FIL screen to identify morphogenetic regulatory genes from heterologous organisms are discussed.

Mol Cell Biol, 1997 Nov, 17(11), 6236 - 45
Elimination of defective alpha-factor pheromone receptors; Jenness DD et al.; This report compares trafficking routes of a plasma membrane protein that was misfolded either during its synthesis or after it had reached the cell surface . A temperature-sensitive mutant form of the yeast alpha-factor pheromone receptor (ste2-3) was found to provide a model substrate for quality control of plasma membrane proteins . We show for the first time that a misfolded membrane protein is recognized at the cell surface and rapidly removed . When the ste2-3 mutant cells were cultured continuously at 34 degrees C, the mutant receptor protein (Ste2-3p) failed to accumulate at the plasma membrane and was degraded with a half-life of 4 min, compared with a half-life of 33 min for wild-type receptor protein (Ste2p) . Degradation of both Ste2-3p and Ste2p required the vacuolar proteolytic activities controlled by the PEP4 gene . At 34 degrees C, Ste2-3p comigrated with glycosylated Ste2p on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that Ste2-3p enters the secretory pathway . Degradation of Ste2-3p did not require delivery to the plasma membrane as the sec1 mutation failed to block rapid turnover . Truncation of the C-terminal cytoplasmic domain of the mutant receptors did not permit accumulation at the plasma membrane; thus, the endocytic signals contained in this domain are unnecessary for intracellular retention . In the pep4 mutant, Ste2-3p accumulated as series of high-molecular-weight species, suggesting a potential role for ubiquitin in the elimination process . When ste2-3 mutant cells were cultured continuously at 22 degrees C, Ste2-3p accumulated in the plasma membrane . When the 22 degrees C culture was shifted to 34 degrees C, Ste2-3p was removed from the plasma membrane and degraded by a PEP4-dependent mechanism with a 24-min half-life; the wild-type Ste2p displayed a 72-min half-life . Thus, structural defects in Ste2-3p synthesized at 34 degrees C are recognized in transit to the plasma membrane, leading to rapid degradation, and Ste2-3p that is preassembled at the plasma membrane is also removed and degraded following a shift to 34 degrees C.

Mol Cell Biol, 1997 Nov, 17(11), 6212 - 22
Role for ADA/GCN5 products in antagonizing chromatin-mediated transcriptional repression; Pollard KJ et al.; The Saccharomyces cerevisiae SWI/SNF complex is a 2-MDa multimeric assembly that facilitates transcriptional enhancement by antagonizing chromatin-mediated transcriptional repression . We show here that mutations in ADA2, ADA3, and GCN5, which are believed to encode subunits of a nuclear histone acetyltransferase complex, cause phenotypes strikingly similar to that of swi/snf mutants . ADA2, ADA3, and GCN5 are required for full expression of all SWI/SNF-dependent genes tested, including HO, SUC2, INO1, and Ty elements . Furthermore, mutations in the SIN1 gene, which encodes a nonhistone chromatin component, or mutations in histone H3 or H4 partially alleviate the transcriptional defects caused by ada/gcn5 or swi/snf mutations . We also find that ada2 swi1, ada3 swi1, and gcn5 swi1 double mutants are inviable and that mutations in SIN1 allow viability of these double mutants . We have partially purified three chromatographically distinct GCN5-dependent acetyltransferase activities, and we show that these enzymes can acetylate both histones and Sin1p . We propose a model in which the ADA/GCN5 and SWI/SNF complexes facilitate activator function by acting in concert to disrupt or modify chromatin structure.

Arch Biochem Biophys, 1997 Oct 15, 346(2), 269 - 76
Distal site and surface mutations of cytochrome P450 1A2 markedly enhance dehalogenation of chlorinated hydrocarbons; Yanagita K et al.; Chlorinated compounds such as chlorinated ethylenes and ethanes are serious environmental pollutants . In the present study, we examined whether or not a recombinant strain of Saccharomyces cerevisiae that expresses rat liver cytochrome P450 1A2 (P450 1A2) wild-type and mutant proteins can efficiently catalyze oxidative and reductive dehalogenations of trichloroethylene, pentachloroethane, and hexachloroethane . Mutations at putative heme distal and protein surface sites of P450 1A2 greatly enhanced turnover values toward those substrates under both aerobic and anaerobic conditions . For example, a Thr319Ala mutation at the putative heme distal site enhanced the degradation rate of trichloroethylene and pentachloroethane by 2- and 2.7-fold, respectively, under aerobic conditions . The Thr319Ala mutation also strongly facilitated the reaction with hexachloroethane up to 13- and 4.5-fold under aerobic and anaerobic conditions, respectively . The Thr319Ala mutation increased dechlorinated over protonated product ratios by 3-fold as well when either pentachloroethane or hexachloroethane was used as a substrate . A Lys250Leu mutation on the putative protein surface site enhanced the dehalogenation rate of hexachloroethane up to 4.8-fold under anaerobic conditions . In contrast, a Glu318Ala mutation at the putative distal site markedly decreased the activities with trichloroethylene and pentachloroethane substrates under aerobic conditions . Conserved amino acids Thr319 and Glu318 at the heme distal site have been suggested to be important in the O2 activation during monooxidation reactions of P450s . However, the present study indicates that Thr319 is likely to be an inhibitor of dechlorination of trichloroethylene and penta- and hexachloroethanes . The roles of Thr319, Glu318, and Lys250 in the catalysis with chlorinated hydrocarbons are discussed in association with reaction mechanisms.

Arch Biochem Biophys, 1997 Oct 15, 346(2), 219 - 29
Nitrobenzimidazoles as substrates for DT-diaphorase and redox cycling compounds: their enzymatic reactions and cytotoxicity; Sarlauskas J et al.; We have synthesized a number of nitrobenzimidazoles containing nitro groups in the benzene ring and found that they acted as relatively efficient substrates for rat liver DT-diaphorase (EC 1.6.99.2), their reactivity exceeding reactivities of nitrofurans and nitrobenzenes . Nitrobenzimidazoles were competitive with NADPH inhibitors of DT-diaphorase in menadione reductase reactions, their inhibition constant being unchanged in the presence of dicumarol and being increased in the presence of 2',5'-ADP . These data indicate that the poor reactivity of nitrobenzimidazoles and other nitroaromatics in comparison to quinones could be determined by their binding in the adenosine-phosphate binding region of the NADPH-binding site, whereas quinones bind at the nicotinamide-binding pocket at the vicinity of FAD of DT-diaphorase . The reduction of 4,5,6-trinitrobenzimidazol-2-one by DT-diaphorase most probably involves reduction of 5-nitro group to 5-nitroso or 5-hydroxylamine derivative at the initial step . A certain parallelism existed between reactivities of nitrobenzimidazoles toward DT-diaphorase and their reactivities in single-electron reduction by Anabaena ferredoxin:NADP+ reductase (EC 1.18.1.2) and Saccharomyces cerevisiae flavocytochrome b2 (EC 1.1.2.3), the latter being determined by electronic factors . However, we suppose that the relatively high reactivity of polinitrobenzimidazoles toward DT-diaphorase was due not only to electronic effects, but also to a sterical crowding of nitrogroups by each other . The toxicity of nitrobenzimidazoles to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) with a moderate amount of DT-diaphorase (260 U/mg protein) is partly prevented by dicumarol . That points out to partial determination of nitrobenzimidazole cytotoxicity by their reduction by DT-diaphorase . Another important factor of nitrobenzimidazole toxicity to this cell line was oxidative stress, catalyzed by single-electron transferring enzymes.

J Virol, 1997 Nov, 71(11), 8552 - 62
Epstein-Barr virus EBNA3C represses Cp, the major promoter for EBNA expression, but has no effect on the promoter of the cell gene CD21; Radkov SA et al.; EBNA3C is a potent repressor of transcription when bound to DNA as a fusion with the DNA binding domain (DBD) of GALA . A survey of promoters has revealed that the wild-type, unfused EBNA3C can specifically repress expression from reporter plasmids containing the Epstein-Barr virus Cp latency-associated promoter . Repression of Cp activity required amino acids 207 to 368, which encompasses a region resembling a basic DBD adjacent to a leucine zipper DNA binding motif and a site which binds to the cellular factor CBF1/RBP-Jkappa . However, amino acids 207 to 368 are dispensable when the protein is bound to DNA as a fusion with the GAL4 DBD, thus implicating this region in DNA binding . Mutation of the CBF1/RBP-Jkappa binding site in EBNA3C abrogated repression, strongly suggesting that CBF1/RBP-Jkappa is necessary for targeting the viral protein to Cp . Consistent with this result, mutation of the EBNA2 response element (a CBF1/RBP-Jkappa binding site) in Cp also prevented significant repression . In addition, amino acids 346 to 543, which were previously defined as important for the repressor activity of the GAL4-EBNA3C fusion proteins, also appear to be necessary for the repression of Cp . Since repression by these fusions was not observed in all cell types, it seems likely that EBNA3C either depends on a corepressor which may interact with amino acids 346 to 543 or is modified in a cell-specific manner in order to repress . These data are consistent with EBNA3C contributing to the regulation of EBNA expression in latently infected B cells through CBF1/RBP-Jkappa and another factor, but this need not directly involve EBNA2 . Finally, although it has been reported that EBNA3C can upregulate CD21 in some B cells, we were unable to demonstrate any effect of EBNA3C on reporter plasmids which contain the CD21 promoter.

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 11798 - 802
Prp43: An RNA helicase-like factor involved in spliceosome disassembly; Arenas JE et al.; The Saccharomyces cerevisiae genes PRP2, PRP16, and PRP22 encode pre-mRNA splicing factors that belong to the highly conserved "DEAH" family of putative RNA helicases . We previously identified two additional members of this family, JA1 and JA2 . To investigate its biological function, we cloned the JA1 gene and generated alleles carrying mutations identical to those found in highly conserved regions of other members of the DEAH family . A ja1 allele carrying a mutation identical to that in the temperature-sensitive (ts) prp22-1 gene conferred ts phenotype when integrated into the genome of a wild-type strain by gene replacement . Northern analysis of RNA obtained from the ts strain shifted to a nonpermissive temperature revealed accumulation of unspliced pre-mRNAs and excised intron lariats . Furthermore, analysis of splicing complexes showed that intron lariats accumulated in spliceosomes . The results presented indicate that JA1 encodes a pre-mRNA processing factor (Prp) involved in disassembly of spliceosomes after the release of mature mRNA . We have therefore renamed this gene PRP43.

Eur J Biochem, 1997 Sep 15, 248(3), 717 - 23
Palytoxin-induced channel formation within the Na+/K+-ATPase does not require a catalytically active enzyme; Scheiner-Bobis G et al.; It has been demonstrated that palytoxin binds to and forms a channel within the Na+/K+-ATPase . To investigate whether palytoxin-induced channel formation within the sodium pump can occur independently of ATP hydrolysis and phosphorylation of the enzyme, an Asp369-->Ala mutant of the alpha1 subunit of the sheep sodium pump was produced and coexpressed with beta subunits in the yeast Saccharomyces cerevisiae . This aspartic acid residue, which during ion transport becomes phosphorylated from ATP, is essential for the function of the sodium pump . Therefore, as expected, microsomes isolated from yeast expressing the mutant sodium pump do not exhibit any ouabain sensitive ATPase activity, whereas in microsomes from yeast expressing the wild-type sodium pump, 60% of the total ATPase activity is ouabain-sensitive . Ouabain binds to yeast membranes containing either wild-type or mutant sodium pumps with similar Bmax (1.45+/-0.05 versus 1.37+/-0.02 pmol/mg) and Kd values (27.7+/-0.91 versus 29.57+/-0.93 nM), thus indicating that the mutant sodium pumps are expressed in the yeast and that the mutation does not considerably affect the conformation of the enzyme . In the presence of phosphate ouabain binds to microsomes containing the wild-type sodium pump with a Kd of 3.62+/-0.34 nM, showing that, although not necessary, phosphoenzyme formation enhances binding of the steroid . Phosphate or ATP, however, inhibit binding of ouabain to microsomes containing the mutant sodium pump with IC50 values of 78+/-3 microM and 3.0+/-0.4 microM, respectively . Despite these radical changes in the interactions of the mutant enzyme with ouabain, the interactions with palytoxin are not affected by the mutation . Palytoxin causes K+ efflux from yeast cells expressing the wild-type or mutant sodium pumps with EC50 values of 3.5+/-0.4 nM and 6.2+/-0.9 nM, respectively . Palytoxin-induced efflux from cells expressing wild-type or mutant sodium pumps occurs with similar t1/2 values of 20.3+/-2.1 min and 22.2+/-3.1 min, respectively . Ouabain inhibits K+ efflux from both cell types with IC50 values of 28+/-2 microM and 210+/-15 microM, respectively . Cells expressing the Asp369-->Ala mutants have an IC50 7.5-fold higher than that obtained with cells expressing the wild-type sodium pumps, possibly because ATP or phosphate present in the cytosol of the yeast cells influence and decrease ouabain binding to the mutant sodium pump . Thus, while ouabain binding and the associated inhibition of ion fluxes is promoted by phosphorylation of the wild-type enzyme by phosphate or ATP, palytoxin-induced channel formation is independent of phosphorylation and can be separated from the ATPase function of the sodium pump . Since ion fluxes through the sodium pump protein do not depend on ATP hydrolysis, the results suggest that the ionophores of pumps and ion channels might share common structural features.

Mol Gen Genet, 1997 Sep, 256(1), 18 - 27
Trichoderma reesei sequences that bind to the nuclear matrix enhance transformation frequency; Belshaw NJ et al.; Three DNA fragments, trs1, 2 and 3, were isolated from the Trichoderma reesei genome on the basis of their ability to promote autonomous replication of plasmids in Saccharomyces cerevisiae . Each trs element bound specifically to the isolated T . reesei nuclear matrix in vitro, and two of them bound in vivo, indicating that they are matrix attachment regions (MARs) . A similar sequence previously isolated from Aspergillus nidulans (ans1) was also shown to bind specifically to the T . reesei nuclear matrix in vitro . The T . reesei MARs are AT-rich sequences containing 70%, 86% and 73% A + T over 2.9, 0.8 and 3.7 kb, respectively for trs1, 2 and 3 . They exhibited no significant sequence homology, but were shown to contain a number of sequence motifs that occur frequently in many MARs identified in other eukaryotes . However, these motifs occurred as frequently in the trs elements as in randomly generated sequences with the same A + T content . trs1 and 3 were shown to be present as single copies in the T . reesei genome . The presence of the trs elements in transforming plasmids enhanced the frequency of integrative transformation of T . reesei up to five fold over plasmids without a trs . No evidence was obtained to suggest that the trs elements promoted efficient replication of plasmids in T . reseei . A mechanism for the enhancement of transformation frequency by the trs elements is proposed.

J Biol Chem, 1997 Oct 24, 272(43), 27444 - 9
Identification of elongin C sequences required for interaction with the von Hippel-Lindau tumor suppressor protein; Takagi Y et al.; Elongin C is a 112-amino acid protein that is found in mammalian cells as a positive regulatory subunit of heterotrimeric RNA polymerase II elongation factor Elongin (SIII) and as a component of a multiprotein complex containing the von Hippel-Lindau (VHL) tumor suppressor protein . As a subunit of the Elongin complex, Elongin C interacts directly with the transcriptionally active Elongin A subunit and potently induces its elongation activity; in addition, Elongin C interacts with the ubiquitin-like Elongin B subunit, which regulates the interaction of Elongin C with Elongin A . As a component of the VHL complex, Elongin C interacts directly with both Elongin B and the VHL protein . Binding of the VHL protein to Elongin C was found to prevent Elongin C from interacting with and activating Elongin A in vitro, leading to the proposal that one function of the VHL protein may be to regulate RNA polymerase II elongation by negatively regulating the Elongin complex . In this report, we identify Elongin C sequences required for its interaction with the VHL protein . We previously demonstrated that the ability of Elongin C to bind and activate Elongin A is sensitive to mutations in the C-terminal half of Elongin C, as well as to mutations in an N-terminal Elongin C region needed for formation of the Elongin BC complex . Here we show that interaction of Elongin C with the VHL tumor suppressor protein depends strongly on sequences in the C terminus of Elongin C but is independent of the N-terminal Elongin C region required for binding to Elongin B and for binding and activation of Elongin A . Taken together, our results are consistent with the proposal that the VHL protein negatively regulates Elongin C activation of the Elongin complex by sterically blocking the interaction of C-terminal Elongin C sequences with Elongin A . In addition, our finding that only a subset of Elongin C sequences required for its interaction with Elongin A are critical for binding to VHL may offer the opportunity to develop reagents that selectively interfere with Elongin and VHL function.

J Biol Chem, 1997 Oct 24, 272(43), 27281 - 7
Serine phosphorylation-dependent association of the band 4.1-related protein-tyrosine phosphatase PTPH1 with 14-3-3beta protein; Zhang SH et al.; PTPH1 is a human protein-tyrosine phosphatase with homology to the band 4.1 superfamily of cytoskeletal-associated proteins . PTPH1 was found to associate with 14-3-3beta using a yeast two-hybrid screen, and its interaction could be reconstituted in vitro using recombinant proteins . Examination of the interaction between 14-3-3beta and various deletion mutants of PTPH1 by two-hybrid tests suggested that the integrity of the PTP is important for this binding . Although both PTPH1 and Raf-1 form complexes with 14-3-3beta, they appear to do so independently . Binding of 14-3-3beta to PTPH1 in vitro was abolished by pretreating PTPH1 with potato acid phosphatase and was greatly enhanced by pretreating with Cdc25C-associated protein kinase . Thus the association between PTPH1 and 14-3-3beta is phosphorylation-dependent . Two novel motifs RSLS359VE and RVDS853EP in PTPH1 were identified as major 14-3-3beta-binding sites, both of which are distinct from the consensus binding motif RSXSXP recently found in Raf-1 . Mutation of Ser359 and Ser853 to alanine significantly reduced the association between 14-3-3beta and PTPH1 . Furthermore, association of PTPH1 and 14-3-3beta was detected in several cell lines and was regulated in response to extracellular signals . These results raise the possibility that 14-3-3beta may function as an adaptor molecule in the regulation of PTPH1 and may provide a link between serine/threonine and tyrosine phosphorylation-dependent signaling pathways.

J Biol Chem, 1997 Oct 24, 272(43), 27259 - 65
Defining the minimal domain of Ku80 for interaction with Ku70; Osipovich O et al.; The Ku protein has a critical function in the repair of double-strand DNA breaks induced for example by ionizing radiation or during VDJ recombination . Ku serves as the DNA-binding subunit of the DNA-dependent kinase and is a heterodimeric protein composed of 80- and 70-kDa subunits . We used the two-hybrid system to analyze the interaction domains of the Ku subunits and to identify possible additional partners for Ku . Screening a human cDNA library with the Ku heterodimer did not reveal any novel partners . Screening with the individual subunits, we detected only Ku70 clones interacting with Ku80 and only Ku80 clones interacting with Ku70, indicating that these are the primary partners for one another . Ku80 and Ku70 formed only heterodimers and did not homodimerize . Ku80 was restricted to interacting with just one Ku70 molecule at a time . The minimal functional interaction domain of Ku80 that interacted with Ku70 was defined . It consisted of a 28-amino acid region extending from amino acid 449 to 477 . This region was crucial for interaction with Ku70, since mutation within this critical site at amino acids 453 and 454 abrogated the ability to interact with Ku70 . We furthermore verified that the same region is crucial for interaction with Ku70 using in vitro co-translation of both subunits followed by an immunoprecipitation with anti-Ku70 antibodies . This interaction domain of Ku80 does not contain any motif previously recognized in protein-protein interactions.

J Biol Chem, 1997 Oct 24, 272(43), 27253 - 8
The histone acetyltransferase activity of human GCN5 and PCAF is stabilized by coenzymes; Herrera JE et al.; Here we report that PCAF and human GCN5, two related type A histone acetyltransferases, are unstable enzymes that under the commonly used assay conditions are rapidly and irreversibly inactivated . In addition, we report that free histone H1, although not acetylated in vivo, is a preferred and convenient in vitro substrate for the study of PCAF, human GCN5, and possibly other type A histone acetyltransferases . Using either histone H1 or histone H3 as substrates, we find that preincubation with either acetyl-CoA or CoA stabilizes the acetyltransferase activities of PCAF, human GCN5 and an enzymatically active PCAF deletion mutant containing the C-terminal half of the protein . The stabilization requires the continuous presence of coenzyme, suggesting that the acetyltransferase-coenzyme complexes are stable, while the isolated apoenzymes are not . Human GCN5 and the N-terminal deletion mutant of PCAF are stabilized equally well by preincubation with either CoA or acetyl-CoA, while intact PCAF is better stabilized by acetyl-CoA than by CoA . Intact PCAF, but not the N-terminal truncation mutant or human GCN5, is autoacetylated . These findings raise the possibility that the intracellular concentrations of the coenzymes affect the stability and therefore the nuclear activity of these acetyltransferases.

J Biol Chem, 1997 Oct 24, 272(43), 27160 - 6
Functional domain mapping of the clathrin-associated adaptor medium chains mu1 and mu2; Aguilar RC et al.; The clathrin-associated adaptors AP-1 and AP-2 are heterotetrameric complexes involved in the recognition of sorting signals present within the cytosolic domain of integral membrane proteins . The medium chains of these complexes, mu1 and mu2, have been implicated in two types of interaction: assembly with the beta1 and beta2 chains of the corresponding complexes and recognition of tyrosine-based sorting signals . In this study, we report the results of a structure-function analysis of the mu1 and mu2 chains aimed at identifying regions of the molecules that are responsible for each of the two interactions . Analyses using the yeast two-hybrid system and proteolytic digestion experiments suggest that mu1 and mu2 have a bipartite structure, with the amino-terminal one-third (residues 1-145 of mu1 and mu2) being involved in assembly with the beta chains and the carboxyl-terminal two-thirds (residues 147-423 of mu1 and 164-435 of mu2) binding tyrosine-based sorting signals . These observations support a model in which the amino-terminal one-third of mu2 is embedded within the core of the AP-2 complex, while the carboxyl-terminal two-thirds of the protein are exposed to the medium, placing this region in a position to interact with tyrosine-based sorting signals.

J Biol Chem, 1997 Oct 24, 272(43), 26991 - 8
Isolation and characterization of a dual prenylated Rab and VAMP2 receptor; Martincic I et al.; Rab GTPases have been implicated in intracellular vesicle trafficking . Using the yeast two-hybrid screen, we have isolated a rat clone that interacts with Rab3A as well as with Rab1 . The gene encodes a 20.6-kDa protein with two extensive hydrophobic domains and is broadly expressed in all tissues . This protein binds to prenylated Rab GTPases but not to other small Ras-like GTPases such as the Rho/Rac family . This prenylated Rab acceptor (PRA1) also binds specifically to the synaptic vesicle protein VAMP2 (or synaptobrevin II) but shows no affinity for VAMP1 or cellubrevin in both the yeast two-hybrid system and in vitro binding assays . This specificity resides, in part, in the proline-rich domain of VAMP2 as a chimera containing this domain of VAMP2 fused to VAMP1 is able to bind to PRA1 . The transmembrane domain of VAMP2 is also essential as its deletion abolished binding to PRA1 . Replacement of the deleted VAMP2 transmembrane domain by a CAAX prenylation signal can not restore binding to PRA1 . This interaction is therefore distinct from that required for VAMP2 binding to either syntaxin or both syntaxin and SNAP-25 . Deletion analysis on PRA1 indicates that the critical Rab- and VAMP2-interacting residues reside in two regions: the amino-terminal residues 30-54 and the extreme carboxyl-terminal domain . This dual Rab and VAMP2 binding characteristic suggests that PRA1 may serve to link these two protein families in the control of vesicle docking and fusion.

Regul Toxicol Pharmacol, 1997 Aug, 26(1 Pt 1), 60 - 8
Development of a Tier I screening battery for detecting endocrine-active compounds (EACs); Cook JC et al.; One of the components of our research program is development of a mode-of-action screening battery to detect several different types of endocrine-active compounds (EACs) . Our working hypothesis is that a comprehensive short-term in vivo/in vitro battery can be developed to identify endocrine toxicants using a collection of endpoints . The goals of this battery are that it be quick, cost effective, and predictive . The purpose of this battery is to identify potential EACs and to assess their potency in order to prioritize compounds for further study . Two in vivo screens (intact male and ovariectomized female rats) are being evaluated for their ability to detect several different types of endocrine activity . To validate this screen, 15 compounds with known endocrine activities are being used to evaluate a collection of different endpoints for their variability, stability over time, predictiveness, and dose dependency . These positive controls were chosen because they can modulate development, reproduction, or cancer . The advantage of an in vivo screen is that it utilizes a metabolically and physiologically intact system . The male in vivo battery will be used to assess several different types of endocrine activity, primarily by using a comprehensive hormonal battery . The female in vivo battery will be used to identify compounds which are either estrogenic/antiestrogenic or can alter the prolactin pathway . The in vitro portion of the screening battery consists of a yeast transactivation system (YTS) . The YTS is being evaluated for its ability to identify compounds which are agonists or antagonists to the estrogen, androgen, or progesterone receptors . The expression of mammalian receptors in yeast allows for assessment of steroid-dependent transcriptional activators . The value of this system is that it can be used as a routine screen for compounds that interact with steroid receptors . Alterations in ligand binding to these receptors can be correlated with alterations in development via masculinization of females and/or feminization of males, decreases in reproductive success, or modulation of cancer incidence from in vivo tests . The in vivo and in vitro screens are designed to be run in parallel with built-in redundancy in order to reduce the probability of false-negative/ positive responses.

Genomics, 1997 Oct 1, 45(1), 200 - 10
Genomic organization and molecular characterization of a gene encoding HsPXF, a human peroxisomal farnesylated protein; Kammerer S et al.; A protein modification essential for the cellular sorting of many biologically relevant proteins is the covalent attachment of prenyl lipids by specific transferases . Isoprenylation is known to render protein domains hydrophobic, thereby facilitating the interaction with lipid bilayers and/or membrane proteins . The target for the modification with farnesyl groups is the COOH-terminal sequence CaaX . Among the variety of farnesylated proteins the only one reported so far to be located to peroxisomes is the 37-kDa peroxisomal farnesylated hamster protein PxF . Recently we published data on the cDNA of the human gene HK33 (A . Braun et al., 1994, Gene 146: 291-295), which was revealed to be the human ortholog of PxF and was consequently renamed HsPXF . The genomic structure, molecular characterization, and evolutionary conservation of HsPXF are described herein . The exact location of the gene was defined as chromosome 1q22 . The gene spans a region of approximately 9 kb, containing eight exons and seven introns . The 5' upstream region showed two potential Sp1-binding sites and an Alu repetitive sequence . Luciferase reporter activating capacity confirmed the presumed promoter activity of this region . On the transcriptional level, we detected four splice variants originating either from exon skipping or from alternative splicing events . For the HsPXF protein, a carboxyterminal farnesylation at cysteine residues was demonstrated . Through the use of HsPXF-specific antibodies, the protein was shown to be attached to the outer surface of peroxisomes . This localization together with the similarity to a peroxisomal assembly protein from Saccharomyces cerevisiae suggests HsPXF is involved in the process of peroxisomal biogenesis or assembly.

Curr Genet, 1997 Sep, 32(3), 225 - 30
Hydrophobin gene srh1, expressed during sporulation of the biocontrol agent Trichoderma harzianum; Munoz G et al.; A cDNA clone encoding a spore-related hydrophobin, SRHI, was isolated from a cDNA bank prepared from mRNA induced in sporulating cultures of Trichoderma harzianum by heterologous hybridization using the hfb2 gene encoding a spore-bound hydrophobin of Trichoderma reesei as a probe . Based on sequence similarity the predicted protein was identified as a new member of the class-II hydrophobin family . Including the signal sequences, SRHI has 65% and 56% amino-acid similarity with the T . reesei hydrophobins HFBII and HFBI, respectively, being less similar with other hydrophobins . srh1 is present as one copy in the T . harzianum genome . It is highly expressed under sporulating conditions, both in submerged as well as in aerial cultures . Moreover, nutrient limitation induces srh1 expression.

Curr Genet, 1997 Sep, 32(3), 175 - 81
Association of transcripts from a group-I intron-containing gene with high sedimentation coefficient particles; Richard GF et al.; The mitochondrial gene coding for the large rRNA contains a self-splicing optional group-I intron (Sc-LSU.1) in some Saccharomyces cerevisiae strains . Although the mechanisms of splicing have been extensively studied, little is known about the possible interactions of this intron with other mitochondrial molecules such as proteins . Using glycerol gradients, we have compared the sedimentation coefficients of mitochondrial transcripts containing the Sc-LSU.1 intron in native yeast extracts and in purified RNA preparations . By comparing extracts from rho+ and rho- cells we have found that at least three RNA species containing the Sc-LSU.1 intron (4.5 kb, 2.7 kb and 1.2 kb respectively) are associated in vivo with a multimolecular complex of sedimentation coefficient 50S made up of nuclearly encoded proteins . Another RNA species of 2.7 kb, which may correspond to a cleavage at the dodecamer sequence of the intron, is not associated with the same particle . The possibility that the 50S particle corresponds to the mitochondrial ribosome or its precursor form(s) is discussed.

Curr Genet, 1997 Sep, 32(3), 163 - 74
A novel nuclear gene, CBT1, essential for mitochondrial cytochrome b formation: terminal processing of mRNA and intron dependence; Rieger KJ et al.; We describe a new nuclear gene, CBT1 (Cytochrome B Termination), specifically involved in the generation of mature mRNA of cytochrome b in yeast mitochondria . Disruption of CBT1 (corresponding to ORF YKL 208W) results in a respiratory deficiency (no growth on acetate and ethanol, a reduced growth on glycerol, and a moderate growth on lactate) . Cytochrome b is practically undetectable spectrally, while cytochromes a and a3 (cytochrome oxidase) appear unaffected by the disruption . Analysis of mitochondrial transcripts shows a reduced abundance of cytb mRNA, which in addition is approximately 200 nucleotides longer than that of the wild-type . Sequencing of the 3' region of the mutant cytb mRNA with an oligonucleotide primer positioned 148 nt downstream from the dodecamer sequence ("end-of-messenger" signal), demonstrates that the mutant transcript is extended beyond this position and is not processed at the conserved dodecamer cleavage site . The CBT1 gene product may be one of the components required for the exact 3' cleavage of the cytb messenger and may also be related to RNA splicing, since the intron-containing cytb gene is not as well expressed as the intron-less gene and the respiratory deficiency is more severe . We propose, that the CBT1 protein is necessary for the correct trimming of the end of cytb pre-mRNA and may be a part of the multi-component complex involved in this process.

Biochem J, 1997 Aug 15, 326 ( Pt 1), 149 - 57
Phosphorylation of serine-167 on the human oestrogen receptor is important for oestrogen response element binding and transcriptional activation; Castano E et al.; We have studied the role of phosphorylation of the human oestrogen receptor (hOR; otherwise known as hER) at serine-167, which has been identified previously as the major oestrogen-induced phosphorylation site . We have tested transactivation by the hOR in yeast and cell-free transcription assays, and shown that mutation of serine-167 results in a 70% decrease in hOR-dependent transcription . Furthermore we explored the functional significance of phosphorylation at this site by hormone binding and DNA binding . DNA binding affinity was 10-fold lower when serine-167 was changed to alanine in the hOR . Cell-free transcription experiments showed that casein kinase II is the enzyme responsible for oestradiol-dependent phosphorylation of the hOR at serine-167 . This suggests that a conformational change of the hOR must occur upon hormone binding that exposes serine-167 to casein kinase II, resulting in transactivation of oestrogen-responsive genes.

J Cell Biol, 1997 Oct 20, 139(2), 351 - 63
The phosphatidylinositol transfer protein domain of Drosophila retinal degeneration B protein is essential for photoreceptor cell survival and recovery from light stimulation; Milligan SC et al.; The Drosophila retinal degeneration B (rdgB) gene encodes an integral membrane protein involved in phototransduction and prevention of retinal degeneration . RdgB represents a nonclassical phosphatidylinositol transfer protein (PITP) as all other known PITPs are soluble polypeptides . Our data demonstrate roles for RdgB in proper termination of the phototransduction light response and dark recovery of the photoreceptor cells . Expression of RdgB's PITP domain as a soluble protein (RdgB-PITP) in rdgB2 mutant flies is sufficient to completely restore the wild-type electrophysiological light response and prevent the degeneration . However, introduction of the T59E mutation, which does not affect RdgB-PITP's phosphatidylinositol (PI) and phosphatidycholine (PC) transfer in vitro, into the soluble (RdgB-PITP-T59E) or full-length (RdgB-T59E) proteins eliminated rescue of retinal degeneration in rdgB2 flies, while the light response was partially maintained . Substitution of the rat brain PITPalpha, a classical PI transfer protein, for RdgB's PITP domain (PITPalpha or PITPalpha-RdgB chimeric protein) neither restored the light response nor maintained retinal integrity when expressed in rdgB2 flies . Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo . Expression of either RdgB-T59E or PITPalpha-RdgB in rdgB+ flies produced a dominant retinal degeneration phenotype . Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPalpha-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism . This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.

Genes Dev, 1997 Oct 15, 11(20), 2741 - 51
Alternative 3'-end processing of U5 snRNA by RNase III; Chanfreau G et al.; The cellular components required to form the 3' ends of small nuclear RNAs are unknown . U5 snRNA from Saccharomyces cerevisiae is found in two forms that differ in length at their 3' ends (U5L and U5S) . When added to a yeast cell free extract, synthetic pre-U5 RNA bearing downstream genomic sequences is processed efficiently and accurately to generate both mature forms of U5 . The two forms of U5 are produced in vitro by alternative 3'-end processing . A temperature-sensitive mutation in the RNT1 gene encoding RNase III blocks accumulation of U5L in vivo . In vitro, alternative cleavage of the U5 precursor by RNase III determines the choice between the two multistep pathways that lead to U5L and U5S, one of which (U5L) is strictly dependent on RNase III . These results identify RNase III as a trans-acting factor involved in 3'-end formation of snRNA and show how RNase III might regulate alternative RNA processing pathways.

Cancer Res, 1997 Oct 1, 57(19), 4285 - 300
Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents; O'Connor PM et al.; In the present study, we report the characterization of the p53 tumor suppressor pathway in the 60 cell lines of the National Cancer Institute (NCI) anticancer drug screen, as well as correlations between the integrity of this pathway and the growth-inhibitory potency of 123 anticancer agents in this screen . Assessment of p53 status in these lines was achieved through complete p53 cDNA sequencing, measurement of basal p53 protein levels and functional assessment of (a) transcriptional activity of p53 cDNA from each line in a yeast assay, (b) gamma-ray-induced G1 phase cell cycle arrest, and (c) gamma-ray-induced expression of CIP1/WAF1, GADD45, and MDM2 mRNA . Our investigations revealed that p53 gene mutations were common in the NCI cell screen lines: 39 of 58 cell lines analyzed contained a mutant p53 sequence . cDNA derived from almost all of the mutant p53 cell lines failed to transcriptionally activate a reporter gene in yeast, and the majority of mutant p53 lines studied expressed elevated basal levels of the mutant p53 protein . In contrast to most of the wild-type p53-containing lines, cells containing mutant p53 sequence were also deficient in gamma-ray induction of CIP1/WAF1, GADD45, and MDM2 mRNA and the ability to arrest in G1 following gamma-irradiation . Taken together, these assessments provided indications of the integrity of the p53 pathway in the 60 cell lines of the NCI cell screen . These individual p53 assessments were subsequently used to probe a database of growth-inhibitory potency for 123 "standard agents," which included the majority of clinically approved anticancer drugs . These 123 agents have been tested against these lines on multiple occasions, and a proposed mechanism of drug action had previously been assigned to each agent . Our analysis revealed that cells with mutant p53 sequence tended to exhibit less growth inhibition in this screen than the wild-type p53 cell lines when treated with the majority of clinically used anticancer agents: including DNA cross-linking agents, antimetabolites, and topoisomerase I and II inhibitors . Similar correlations were uncovered when we probed this database using most of the other indices of p53 status we assessed in the lines . Interestingly, a class of agents that differed in this respect was the antimitotic agents . Growth-inhibitory activity of these agents tended, in this assay, to be independent of p53 status . Our characterization of the p53 pathway in the NCI cell screen lines should prove useful to researchers investigating fundamental aspects of p53 biology and pharmacology . This information also allows for the large-scale analysis of the more than 60,000 compounds tested against these lines for novel agents that might exploit defective p53 function as a means of preferential toxicity.

Am J Hum Genet, 1997 Oct, 61(4), 862 - 7
The X-linked gene G4.5 is responsible for different infantile dilated cardiomyopathies; D'Adamo P et al.; Barth syndrome (BTHS) is an X-linked disorder characterized clinically by the associated features of cardiac and skeletal myopathy, short stature, and neutropenia . The clinical manifestations of the disease are, in general, quite variable, but cardiac failure as a consequence of cardiac dilatation and hypertrophy is a constant finding and is the most common cause of death in the first months of life . X-linked cardiomyopathies with clinical manifestations similar to BTHS have been reported, and it has been proposed that they may be allelic . We have recently identified the gene responsible for BTHS, in one of the Xq28 genes, G4.5 . In this paper we report the sequence analysis of 11 additional familial cases: 8 were diagnosed as possibly affected with BTHS, and 3 were affected with X-linked dilated cardiomyopathies . Mutations in the G4.5 gene were found in nine of the patients analyzed . The molecular studies have linked together what were formerly considered different conditions and have shown that the G4.5 gene is responsible for BTHS (OMIM 302060), X-linked endocardial fibroelastosis (OMIM 305300), and severe X-linked cardiomyopathy (OMIM 300069) . Our results also suggest that very severe phenotypes may be associated with null mutations in the gene, whereas mutations in alternative portions or missense mutations may give a "less severe" phenotype.

Cancer Res, 1997 Oct 15, 57(20), 4564 - 9
Aclacinomycin A stabilizes topoisomerase I covalent complexes; Nitiss JL et al.; Aclacinomycin A (aclarubicin) is an anthracycline anticancer agent with demonstrated activity against both leukemias and solid tumors . Previous results suggested that a major activity of aclacinomycin A is the inhibition of topoisomerase II catalytic activity . We have applied a yeast system to test whether aclacinomycin A is a topoisomerase II inhibitor in vivo and to test whether we could identify other important targets of this drug . We have found that overexpression of yeast topoisomerase II confers resistance to aclacinomycin A in yeast, consistent with the hypothesis that this drug is a catalytic inhibitor of topoisomerase II . Interestingly, we have also found that in yeast, aclacinomycin A, like camptothecin, stabilizes topoisomerase I cleavage . We carried out biochemical analysis with purified human topoisomerase I and demonstrated that this drug efficiently stabilizes topoisomerase I covalent complexes, indicating that aclacinomycin A represents a novel class of combined topoisomerase I/II inhibitor.

Protein Sci, 1997 Oct, 6(10), 2218 - 26
Oligomerization properties of GCN4 leucine zipper e and g position mutants; Zeng X et al.; Putative intersubunit electrostatic interactions between charged amino acids on the surfaces of the dimer interfaces of leucine zippers (g-e' ion pairs) have been implicated as determinants of dimerization specificity . To evaluate the importance of these ionic interactions in determining the specificity of dimer formation, we constructed a pool of > 65,000 GCN4 leucine zipper mutants in which all the e and g positions are occupied by different combinations of alanine, glutamic acid, lysine, or threonine . The oligomerization properties of these mutants were evaluated based on the phenotypes of cells expressing lambda repressor-leucine zipper fusion proteins . About 90% of the mutants do not form stable homooligomers . Surprisingly, approximately 8% of the mutant sequences have phenotypes consistent with the formation of higher-order (> dimer) oligomers, which can be classified into three types based on sequence features . The oligomerization states of mutants from two of these types were determined by characterizing purified fusion proteins . The Type I mutant behaved as a tetramer under all tested conditions, whereas the Type III mutant formed a variety of higher-order oligomers, depending on the solution conditions . Stable homodimers comprise less than 3% of the pool; several g-e' positions in these mutants could form attractive ion pairs . Putative repulsive ion pairs are not found among the homodimeric mutants . However, patterns of charged residues at the e and g positions do not seem to be sufficient to predict either homodimer or heterodimer formation among the mutants.

Genetics, 1997 Oct, 147(2), 493 - 505
Specialized Rap1p/Gcr1p transcriptional activation through Gcr1p DNA contacts requires Gcr2p, as does hyperphosphorylation of Gcr1p; Zeng X et al.; The multifunctional regulatory factor Rap1p of Saccharomyces cerevisiae accomplishes one of its tasks, transcriptional activation, by complexing with Gcr1p . An unusual feature of this heteromeric complex is its apparent capacity to contact simultaneously two adjacent DNA elements (UASRPG and the CT box, bound specifically by Rap1p and Gcr1p, respectively) . The complex can activate transcription through isolated UASRPG but not CT elements . In promoters that contain both DNA signals its activity is enhanced, provided the helical spacing between the two elements is appropriate; this suggests that at least transient DNA loop formation is involved . We show here that this CT box-dependent augmentation of Rap1p/Gcr1p activation requires the presence of a third protein Gcr2p; the Gcr2- growth defect appears to result from a genome-wide loss of the CT box effect . Interestingly, a hyperphosphorylated form of Gcr1p disappears in delta gcr2 cells but reappears if they harbor a doubly point-mutated GCR1 allele that bypasses the Gcr2- growth defect . Gcr2p therefore appears to induce a conformation change in Gcr1p and/or stimulate its hyperphosphorylation; one or both of these effects can be mimicked in the absence of GCR2 by mutation of GCR1 . This improved view of Rap1p/Gcr1p/Gcr2p function reveals a new aspect of eukaryotic gene regulation: modification of an upstream activator, accompanied by at least transient DNA loop formation, mediates its improved capacity to activate transcription.

Biochemistry, 1997 Oct 21, 36(42), 13095 - 101
Azatoxin is a mechanistic hybrid of the topoisomerase II-targeted anticancer drugs etoposide and ellipticine; Cline SD et al.; One approach to broadening the diversity of topoisomerase II-targeted anticancer agents is to generate novel compounds by combining structural elements of drugs known to stimulate enzyme-mediated DNA cleavage . The first agent to emerge from such a rational drug design is azatoxin, a hybrid drug that fuses chemical structures from etoposide and ellipticine . Since these drugs differ significantly in their structural and mechanistic attributes, azatoxin may preferentially retain the functional properties of one of these two drugs, behave as a hybrid molecule, or act as a novel pharmacophore . Therefore, the properties of azatoxin were characterized to determine relationships between its mechanism of action and those of its parent compounds . Azatoxin, like etoposide, binds to DNA in a nonintercalative fashion . However, similar to ellipticine, the drug has no effect on enzyme-mediated DNA religation and apparently stimulates scission primarily by enhancing cleavage complex formation . Depending on the species of topoisomerase II examined, the cleavage potency of azatoxin resembles that of either of its chemical parents . Furthermore, out of 43 DNA cleavage sites analyzed, approximately 90% of those induced by azatoxin are shared with either etoposide, ellipticine, or both drugs . Finally, competition studies indicate that azatoxin interacts with topoisomerase II in the enzyme domain utilized by etoposide and ellipticine . Taken together, these results strongly suggest that azatoxin is a mechanistic hybrid of its parent compounds and shares functional properties with both drugs.

Cell, 1997 Oct 3, 91(1), 35 - 45
Cohesins: chromosomal proteins that prevent premature separation of sister chromatids; Michaelis C et al.; Cohesion between sister chromatids opposes the splitting force exerted by microtubules, and loss of this cohesion is responsible for the subsequent separation of sister chromatids during anaphase . We describe three chromosmal proteins that prevent premature separation of sister chromatids in yeast . Two, Smc1p and Smc3p, are members of the SMC family, which are putative ATPases with coiled-coil domains . A third protein, which we call Scc1p, binds to chromosomes during S phase, dissociates from them at the metaphase-to-anaphase transition, and is degraded by the anaphase promoting complex . Association of Scc1p with chromatin depends on Smc1p . Proteins homologous to Scc1p exist in a variety of eukaryotic organisms including humans . A common cohesion apparatus might be used by all eukaryotic cells during both mitosis and meiosis.

Genes Dev, 1997 Oct 15, 11(20), 2701 - 14
Mammalian homologs of seven in absentia regulate DCC via the ubiquitin-proteasome pathway; Hu G et al.; DCC (deleted in colorectal cancer) is postulated to function as transmembrane receptor for the axon and cell guidance factor netrin-1 . We report here that the DCC cytoplasmic domain binds to proteins encoded by mammalian homologs of the Drosophila seven in absentia (sina) gene, as well as Drosophila Sina . Sina has a critical role in R7 photoreceptor development and shows upward of 85% amino acid identity with its mammalian homologs (termed Siahs), but the function of the Sina/Siah proteins has not been defined . We sought, therefore, to characterize further their interaction with DCC . Immunofluorescence studies suggested the Sina/Siah proteins localized predominantly in the cytoplasm and in association with DCC . DCC was found to be ubiquitinated and the Sina/Siah proteins regulated its expression . Proteasome inhibitors blocked the effects of Sina/Siah on DCC, and the Sina/Siah proteins interacted with ubiquitin-conjugating enzymes (Ubcs) . A mutant Siah protein lacking the amino-terminal Ubc-binding sequences complexed with DCC, but did not degrade it . The in vivo interaction between Sina/Siah and DCC was confirmed through studies of transgenic Drosophila lines in which DCC and Sina were ectopically expressed in the eye . Taken together, the data imply that the Sina/Siah proteins regulate DCC and perhaps other proteins via the ubiquitin-proteasome pathway.

Science, 1997 Oct 17, 278(5337), 460 - 3
CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis; Visintin R et al.; Proteolysis mediated by the anaphase-promoting complex (APC) triggers chromosome segregation and exit from mitosis, yet its regulation is poorly understood . The conserved Cdc20 and Cdh1 proteins were identified as limiting, substrate-specific activators of APC-dependent proteolysis . CDC20 was required for the degradation of the APC substrate Pds1 but not for that of other APC substrates, such as Clb2 and Ase1 . Conversely, cdh1Delta mutants were impaired in the degradation of Ase1 and Clb2 but not in that of Pds1 . Overexpression of either CDC20 or CDH1 was sufficient to induce APC-dependent proteolysis of the appropriate target in stages of the cell cycle in which substrates are normally stable.

Science, 1997 Oct 17, 278(5337), 455 - 60
Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase; Verma R et al.; G1 cyclin-dependent kinase (Cdk)-triggered degradation of the S-phase Cdk inhibitor Sic1p has been implicated in the transition from G1 to S phase in the cell cycle of budding yeast . A multidimensional electrospray mass spectrometry technique was used to map G1 Cdk phosphorylation sites in Sic1p both in vitro and in vivo . A Sic1p mutant lacking three Cdk phosphorylation sites did not serve as a substrate for Cdc34p-dependent ubiquitination in vitro, was stable in vivo, and blocked DNA replication . Moreover, purified phosphoSic1p was ubiquitinated in cyclin-depleted G1 extract, indicating that a primary function of G1 cyclins is to tag Sic1p for destruction . These data suggest a molecular model of how phosphorylation and proteolysis cooperate to bring about the G1/S transition in budding yeast.

Gene, 1997 Sep 15, 197(1-2), 269 - 76
Specific isolation of human rDNA genes by TAR cloning; Kouprina N et al.; Selective cloning of human DNA in YACs from monochromosomal human/rodent hybrid cells lines and radiation hybrids can be accomplished by transformation-associated recombination (TAR) between Alu-containing vector(s) and human DNA in yeast . We have expanded this approach to the specific isolation of repetitive genes from the human genome . Highly selective isolation of human rDNA was accomplished using total human DNA and a pair of differentially marked linear TAR cloning vectors where one contained a small fragment of a human rDNA repeat and the other had an Alu repeat as targeting sequences . About half the transformants that acquired both vectors markers had YACs with human rDNA inserts . These results suggest that TAR can be applied to the general isolation of gene families and amplified region from genomic DNAs.

Biochem Biophys Res Commun, 1997 Sep 29, 238(3), 712 - 6
Down-regulation of Ku autoantigen, DNA-dependent protein kinase, and poly(ADP-ribose) polymerase during cellular senescence; Salminen A et al.; During aging and cellular senescence mutations accumulate in genomic and mitochondrial DNA . Ku autoantigens, DNA-dependent protein kinase, and poly (ADP-ribose) polymerase have an essential role in DNA damage recognition . Our purpose was to find out whether cellular senescence of fibroblasts affects the protein components that recognize DNA damage and induce the repair process . We compared presenescent and replicatively senescent human WI-38 fibroblasts with each other and with SV-40 immortalized and serum-deficient quiescent WI-38 cells . Our results showed that replicative senescence significantly decreased the nuclear level of both p70 and p86 components of Ku autoantigen . SV-40 immortalization and cellular quiescence did not affect the level of the p86 component but slightly increased that of p70 . Both replicative senescence and cellular quiescence decreased the activity of DNA-dependent protein kinase in WI-38 fibroblasts . On the other hand, SV-40 immortalization increased the activity of DNA-dependent protein kinase . The protein level of poly(ADP-ribose) polymerase (PARP) was strongly decreased in replicatively senescent fibroblasts . Quiescence of early-passage fibroblasts also slightly reduced the protein level of PARP . Apoptosis was not observed in replicatively senescent fibroblasts . Our results show that replicative senescence and to some extent cellular quiescence down-regulate the recognition system of DNA damage involving Ku autoantigens, DNA-dependent protein kinase, and PARP and hence could enhance the accumulation of DNA damage during aging.

Cell, 1997 Sep 19, 90(6), 1149 - 59
A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function; Horiuchi H et al.; The small GTPase Rab5 plays an essential role in endocytic traffic . Rab GDP dissociation inhibitor delivers Rab5 to the membrane, where a nucleotide exchange activity allows recruitment of an effector protein, Rabaptin-5 . Here we uncovered a novel 60 kDa Rab5-binding protein, Rabex-5 . Rabex-5 forms a tight physical complex with Rabaptin-5, and this complex is essential for endocytic membrane fusion . Sequencing of mammalian Rabex-5 by nanoelectrospray mass spectrometry and cloning revealed striking homology to Vps9p, a yeast protein implicated in endocytic traffic . Rabex-5 displays GDP/GTP exchange activity on Rab5 upon delivery of the GTPase to the membrane . This demonstrates that a soluble exchange factor coupled to a Rab effector translocates from cytosol to the membrane, where the complex stabilizes the GTPase in the active state.

Cell, 1997 Sep 19, 90(6), 1137 - 48
Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI; Scales SJ et al.; Exocytic transport from the endoplasmic reticulum (ER) to the Golgi complex has been visualized in living cells using a chimera of the temperature-sensitive glycoprotein of vesicular stomatitis virus and green fluorescent protein (ts-G-GFP{ct}) . Upon shifting to permissive temperature, ts-G-GFP(ct) concentrates into COPII-positive structures close to the ER, which then build up to form an intermediate compartment or transport complex, containing ERGIC-53 and the KDEL receptor, where COPII is replaced by COPI . These structures appear heterogenous and move in a microtubule-dependent manner toward the Golgi complex . Our results suggest a sequential mode of COPII and COPI action and indicate that the transport complexes are ER-to-Golgi transport intermediates from which COPI may be involved in recycling material to the ER.

Cell, 1997 Sep 19, 90(6), 1123 - 35
Interhomolog bias during meiotic recombination: meiotic functions promote a highly differentiated interhomolog-only pathway; Schwacha A et al.; Meiotic recombination occurs preferentially between homologous nonsister chromatids rather than between sisters, opposite to the bias of mitotic recombinational repair . We have examined formation of joint molecule recombination intermediates (JMs) between homologs and between sisters in yeast strains lacking the meiotic chromosomal protein Red1, the meiotic recA homolog Dmc1, and/or mitotic recA homolog(s), Rad51, Rad55, and Rad57 . Mutant phenotypes imply that most meiotic recombination occurs via an interhomolog-only pathway along which interhomolog bias is established early, prior to or during double strand break (DSB) formation, and then enforced, just at the time when DSBs initiate JM formation . A parallel, less differentiated pathway yields intersister and, probably, a few interhomolog events . Coordinate action of mitotic recA homologs as one functional unit, two functions of RED1, and an interhomolog interaction function of DMC1 are also revealed.

Cell, 1997 Sep 19, 90(6), 1031 - 9
The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response; Sidrauski C et al.; The endoplasmic reticulum (ER) communicates with the nucleus through the unfolded protein response (UPR), which senses accumulation of unfolded proteins in the ER lumen and leads to increased transcription of genes encoding ER-resident chaperones . As a key regulatory step in this signaling pathway, the mRNA encoding the UPR-specific transcription factor Hac1p becomes spliced by a unique mechanism that requires tRNA ligase but not the spliceosome . Splicing is initiated upon activation of Ire1p, a transmembrane kinase that lies in the ER and/or inner nuclear membrane . We show that Ire1p is a bifunctional enzyme: in addition to being a kinase, it is a site-specific endoribonuclease that cleaves HAC1 mRNA specifically at both splice junctions . The addition of purified tRNA ligase completes splicing; we therefore have reconstituted HAC1 mRNA splicing in vitro from purified components.

Proc Int Conf Intell Syst Mol Biol, 1997, 5, 153 - 6
Identifying chimerism in proteins using hidden Markov models of codon usage; Hunter L et al.; Protein chimerism is a phenomenon involving the combination of multiple ancestral sequences into a single, multi-domain protein through evolution . We propose a novel method for detecting chimeric proteins by analyzing their nucleotide sequence . The method tests for differences in the distributions of synonymous (isoaccepting) codons in different regions of the protein . The test involves the comparison of the ability of varying size hidden Markov models (HMMs) of codon usage to fit the natural sequence, relative to a set of randomized controls . We demonstrate the method on the families of yeast nuclear and mitochondrial amino-acyl tRNA synthetases . The method is potentially useful for the automated screening of entire genomes or large databases.

Proc Int Conf Intell Syst Mol Biol, 1997, 5, 88 - 91
Density of states, metastable states, and saddle points exploring the energy landscape of an RNA molecule; Cupal J et al.; Detailed knowledge of the energy landscape of a biopolymer molecule is a prerequisite for understanding its folding kinetics and its final spatial structure . In the case of RNA we consider the energy landscape defined on the set of all secondary structures that can be formed by a given sequence . We show that the exploration of this energy landscapes is computationally feasible . For this purpose we present a recursive algorithm for computing the complete density of states and discuss its application to tRNA sequences . For shorter sequences a more detailed analysis of the energy surface is possible using a complete list of all secondary structures . In this case we identify metastable states and the saddle points that connect them.

Nucleic Acids Res, 1997 Oct 15, 25(20), 4055 - 60
A conserved core element is functionally important for maize mitochondrial promoter activity in vitro; Caoile AG et al.; We have previously used a homologous in vitro transcription system to define functional elements of the maize mitochondrial atpA promoter . These elements comprise a central domain extending from -7 to +5, relative to the transcription start site, and an upstream domain of 1-3 bp that is purine rich and centered around positions -11 to -12 . Within the central domain lies an essential 5 bp core element . These elements are conserved in many mitochondrial promoters, but their functionality has only been tested for atpA . In this study we have introduced mutations into the corresponding elements of two cox3 promoters and show that while the core element is essential for cox3 promoter activity, upstream element mutations have little or no effect . To define the minimal sequence required for in vitro promoter activity a series of short cloned oligonucleotides corresponding to the atpA promoter was used . While some activity was seen with a 14 bp sequence, full activity required 26 bp, suggesting that elements other than the core and upstream region can influence promoter strength . Another series of clones showed that altered spacing between the upstream and core elements of atpA had a significant effect on promoter activity . These results further define important features of the plant mitochondrial transcriptional machinery.

Mol Cell Biol, 1997 Oct, 17(10), 6139 - 46
VP16 targets an amino-terminal domain of HCF involved in cell cycle progression; Wilson AC et al.; The herpes simplex virus (HSV) regulatory protein VP16 activates HSV immediate-early gene transcription through formation of a multiprotein-DNA complex on viral promoters that includes the preexisting nuclear proteins HCF and Oct-1 . The HCF protein is a complex of amino- and carboxy-terminal polypeptides derived from a large (approximately 2,000-amino-acid) precursor by proteolytic processing . Here we show that a 361-residue amino-terminal region of HCF is sufficient to bind VP16, stabilize VP16-induced complex assembly with Oct-1 and DNA, and activate transcription in vivo . This VP16 interaction region contains six kelch-like repeats, a degenerate repeat motif that is likely to fold as a distinctive beta-propeller structure . The third HCF kelch repeat includes a proline residue (P134) that is mutated to serine in hamster tsBN67 cells, resulting in a temperature-sensitive defect in cell proliferation . This missense mutation also prevents direct association between HCF and VP16, suggesting that VP16 mimics a cellular factor required for cell proliferation . Rescue of the tsBN67 cell proliferation defect by HCF, however, requires both the VP16 interaction domain and an adjacent basic region, indicating that HCF utilizes multiple regions to promote cell cycle progression.

Mol Cell Biol, 1997 Oct, 17(10), 6122 - 30
Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively; Johnson AW; XRN1 encodes an abundant cytoplasmic exoribonuclease, Xrn1p, responsible for mRNA turnover in yeast . A screen for bypass suppressors of the inviability of xrn1 ski2 double mutants identified dominant alleles of RAT1, encoding an exoribonuclease homologous with Xrn1p . These RAT1 alleles restored XRN1-like functions, including cytoplasmic RNA turnover, wild-type sensitivity to the microtubule-destabilizing drug benomyl, and sporulation . The mutations were localized to a region of the RAT1 gene encoding a putative bipartite nuclear localization sequence (NLS) . Fusions to green fluorescent protein were used to demonstrate that wild-type Rat1p is localized to the nucleus and that the mutant alleles result in mislocalization of Rat1p to the cytoplasm . Conversely, targeting Xrn1p to the nucleus by the addition of the simian virus 40 large-T-antigen NLS resulted in complementation of the temperature sensitivity of a rat1-1 strain . These results indicate that Xrn1p and Rat1p are functionally interchangeable exoribonucleases that function in and are restricted to the cytoplasm and nucleus, respectively . It is likely that the higher eukaryotic homologs of these proteins will function similarly in the cytoplasm and nucleus.

Mol Cell Biol, 1997 Oct, 17(10), 6114 - 21
Rnr4p, a novel ribonucleotide reductase small-subunit protein; Wang PJ et al.; Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides . Eukaryotic ribonucleotide reductases are alpha2beta2 tetramers; each of the larger, alpha subunits possesses binding sites for substrate and allosteric effectors, and each of the smaller, beta subunits contains a binuclear iron complex . The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity . Previous work has identified two genes in the yeast Saccharomyces cerevisiae, RNR1 and RNR3, that encode alpha subunits and one gene, RNR2, that encodes a beta subunit . Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the beta-subunit proteins . The phenotype of rnr4 mutants is consistent with that expected for a defect in ribonucleotide reductase; rnr4 mutants are supersensitive to the ribonucleotide reductase inhibitor hydroxyurea and display an S-phase arrest at their restrictive temperature . rnr4 mutant extracts are deficient in ribonucleotide reductase activity, and this deficiency can be remedied by the addition of exogenous Rnr4p . As is the case for the other RNR genes, RNR4 is induced by agents that damage DNA . However, Rnr4p lacks a number of sequence elements thought to be essential for iron binding, and mutation of the critical tyrosine residue does not affect Rnr4p function . These results suggest that Rnr4p is catalytically inactive but, nonetheless, does play a role in the ribonucleotide reductase complex.

Mol Cell Biol, 1997 Oct, 17(10), 6097 - 104
The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination; Kooistra R et al.; The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA . Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described . The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals . DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue . To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis . DmRADS4-deficient flies develop normally, but the females are sterile . Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development . The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate . Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test . These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism . The results also indicate that the recombinational repair pathway is functionally conserved in evolution.

Mol Cell Biol, 1997 Oct, 17(10), 6087 - 96
hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks; Maser RS et al.; We previously identified a conserved multiprotein complex that includes hMre11 and hRad50 . In this study, we used immunofluorescence to investigate the role of this complex in DNA double-strand break (DSB) repair . hMre11 and hRad50 form discrete nuclear foci in response to treatment with DSB-inducing agents but not in response to UV irradiation . hMre11 and hRad50 foci colocalize after treatment with ionizing radiation and are distinct from those of the DSB repair protein, hRad51 . Our data indicate that an irradiated cell is competent to form either hMre11-hRad50 foci or hRad51 foci, but not both . The multiplicity of hMre11 and hRad50 foci is much higher in the DSB repair-deficient cell line 180BR than in repair-proficient cells . hMre11-hRad50 focus formation is markedly reduced in cells derived from ataxia-telangiectasia patients, whereas hRad51 focus formation is markedly increased . These experiments support genetic evidence from Saccharomyces cerevisiae indicating that Mre11-Rad50 have roles distinct from that of Rad51 in DSB repair . Further, these data indicate that hMre11-hRad50 foci form in response to DNA DSBs and are dependent upon a DNA damage-induced signaling pathway.

Mol Cell Biol, 1997 Oct, 17(10), 6029 - 39
CA150, a nuclear protein associated with the RNA polymerase II holoenzyme, is involved in Tat-activated human immunodeficiency virus type 1 transcription; Sune C et al.; Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR . We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors . Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation . The sequence of CA150 contains an extensive glutamine- and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster . Immunodepletion of CA150 abolished Tat trans activation in vitro . Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation . Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II holoenzyme . Furthermore, we found that functional Tat associates with the holoenzyme whereas activation-deficient Tat mutants do not . Thus, we propose that Tat action is transduced via an RNA polymerase II holoenzyme that contains CA150.

Mol Cell Biol, 1997 Oct, 17(10), 6023 - 8
Residues in the WD repeats of Tup1 required for interaction with alpha2; Komachi K et al.; The yeast transcriptional repressor Tup1 contains seven WD repeats which interact with the DNA-binding protein alpha2 . We have identified mutations in Tup1 that disrupt this interaction . The positions of the amino acids changed by these mutations are consistent with Tup1 being folded into a seven-bladed propeller like that formed by another WD repeat-containing protein, the beta subunit of the heterotrimeric G protein used in signal transduction . Our results also indicate that the interaction between Tup1 and alpha2 resembles the interaction between Gbeta and G alpha, suggesting that a similar structural interface is formed by WD repeat proteins that are used in both transcriptional regulation and signal transduction.

Mol Cell Biol, 1997 Oct, 17(10), 5707 - 18
Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain; Chen T et al.; Sam68 is a member of a growing family of proteins that contain a single KH domain embedded in a larger conserved domain of approximately 170 amino acids . Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other KH domains and contain conserved residues . KH domains are protein motifs that are involved in RNA binding and are often present in multiple copies . Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association . Deletion studies demonstrated that the Sam68 KH domain loops 1 and 4 were required for self-association . The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system . In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo . These Sam68 complexes bound homopolymeric RNA and the SH3 domains of p59fyn and phospholipase Cgamma1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer . The formation of the Sam68 complex was inhibited by p59fyn, suggesting that tyrosine phosphorylation regulates Sam68 oligomerization . Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized . In addition, Sam68, GRP33, GLD-1, and Qk1 associated with other KH domain proteins such as Bicaudal C . These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.

Oncogene, 1997 Sep, 15(11), 1289 - 94
Characterization of a highly conserved gene (OS4) amplified with CDK4 in human sarcomas; Su YA et al.; Amplification and overexpression of genes involved in cellular growth control occur frequently in human cancers . Here, we report characterization of the full length OS4 cDNA derived from 12q13-q15 (Su et al., Proc . Natl . Acad . Sci . USA, 91: 9121-9125, 1994), a region frequently amplified in sarcomas and brain tumors . This cDNA consists of 4833 base pairs (bp) encoding an open reading frame (ORF) of 283 amino acids . The ORF predicts a water-soluble acidic (pI 5.50) polypeptide with a molecular weight of 31759 . Database searches revealed highly significant similarity between OS4 and eight proteins predicted from genomic sequences of Caenorhabditis elegans, Schizosaccaharomyces pombe, and Saccharomyces cerevisiae . Thus, OS4 defines a novel evolutionarily conserved gene superfamily . Northern and database analyses revealed OS4 transcripts in numerous human tissues demonstrating its ubiquitous expression . We also observed overexpression of OS4 in three cancer cell lines with amplification of this gene . Furthermore, we detected OS4 amplification in 5/5 primary sarcomas with known amplification of the closely linked marker CDK4 . These results demonstrate that the highly conserved OS4 gene is frequently included in the 12q13-q15 amplicon and may contribute to the development of a subset of sarcomas.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 11102 - 7
Regulation of sulfur assimilation in higher plants: a sulfate transporter induced in sulfate-starved roots plays a central role in Arabidopsis thaliana; Takahashi H et al.; Proton/sulfate cotransporters in the plasma membranes are responsible for uptake of the environmental sulfate used in the sulfate assimilation pathway in plants . Here we report the cloning and characterization of an Arabidopsis thaliana gene, AST68, a new member of the sulfate transporter gene family in higher plants . Sequence analysis of cDNA and genomic clones of AST68 revealed that the AST68 gene is composed of 10 exons encoding a 677-aa polypeptide (74.1 kDa) that is able to functionally complement a Saccharomyces cerevisiae mutant lacking a sulfate transporter gene . Southern hybridization and restriction fragment length polymorphism mapping confirmed that AST68 is a single-copy gene that maps to the top arm of chromosome 5 . Northern hybridization analysis of sulfate-starved plants indicated that the steady-state mRNA abundance of AST68 increased specifically in roots up to 9-fold by sulfate starvation . In situ hybridization experiments revealed that AST68 transcripts were accumulated in the central cylinder of sulfate-starved roots, but not in the xylem, endodermis, cortex, and epidermis . Among all the structural genes for sulfate assimilation, sulfate transporter (AST68), APS reductase (APR1), and serine acetyltransferase (SAT1) were inducible by sulfate starvation in A . thaliana . The sulfate transporter (AST68) exhibited the most intensive and specific response in roots, indicating that AST68 plays a central role in the regulation of sulfate assimilation in plants.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 11079 - 84
Molecular and functional characterization of a novel low-affinity cation transporter (LCT1) in higher plants; Schachtman DP et al.; The transport of cations across membranes in higher plants plays an essential role in many physiological processes including mineral nutrition, cell expansion, and the transduction of environmental signals . In higher plants the coordinated expression of transport mechanisms is essential for specialized cellular processes and for adaptation to variable environmental conditions . To understand the molecular basis of cation transport in plant roots, a Triticum aestivum cDNA library was used to complement a yeast mutant deficient in potassium (K+) uptake . Two genes were cloned that complemented the mutant: HKT1 and a novel cDNA described in this report encoding a cation transporter, LCT1 (low-affinity cation transporter) . Analysis of the secondary structure of LCT1 suggests that the protein contains 8-10 transmembrane helices and a hydrophilic amino terminus containing sequences enriched in Pro, Ser, Thr, and Glu (PEST) . The transporter activity was assayed using radioactive isotopes in yeast cells expressing the cDNA . LCT1 mediated low-affinity uptake of the cations Rb+ and Na+, and possibly allowed Ca2+ but not Zn2+ uptake . LCT1 is expressed in low abundance in wheat roots and leaves . The precise functional role of this cation transporter is not known, although the competitive inhibition of cation uptake by Ca2+ has parallels to whole plant and molecular studies that have shown the important role of Ca2+ in reducing Na+ uptake and ameliorating Na+ toxicity . The structure of this higher plant ion transport protein is unique and contains PEST sequences.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 11055 - 60
Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator; Travis SM et al.; cAMP-dependent phosphorylation activates the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelia . However, the protein phosphatase (PP) that dephosphorylates and inactivates CFTR in airway and intestinal epithelia, two major sites of disease, is not certain . We found that in airway and colonic epithelia, neither okadaic acid nor FK506 prevented inactivation of CFTR when cAMP was removed . These results suggested that a phosphatase distinct from PP1, PP2A, and PP2B was responsible . Because PP2C is insensitive to these inhibitors, we tested the hypothesis that it regulates CFTR . We found that PP2Calpha is expressed in airway and T84 intestinal epithelia . To test its activity on CFTR, we generated recombinant human PP2Calpha and found that it dephosphorylated CFTR and an R domain peptide in vitro . Moreover, in cell-free patches of membrane, addition of PP2Calpha inactivated CFTR Cl- channels; reactivation required readdition of kinase . Finally, coexpression of PP2Calpha with CFTR in epithelia reduced the Cl- current and increased the rate of channel inactivation . These results suggest that PP2C may be the okadaic acid-insensitive phosphatase that regulates CFTR in human airway and T84 colonic epithelia . It has been suggested that phosphatase inhibitors could be of therapeutic value in cystic fibrosis; our data suggest that PP2C may be an important phosphatase to target.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10774 - 9
Substrate specificity determinants in the farnesyltransferase beta-subunit; Trueblood CE et al.; Protein prenyltransferases catalyze the covalent attachment of isoprenoid lipids (farnesyl or geranylgeranyl) to a cysteine near the C terminus of their substrates . This study explored the specificity determinants for interactions between the farnesyltransferase of Saccharomyces cerevisiae and its protein substrates . A series of substitutions at amino acid 149 of the farnesyltransferase beta-subunit were tested in combination with a series of substitutions at the C-terminal amino acid of CaaX protein substrates Ras2p and a-factor . Efficient prenylation was observed when oppositely charged amino acids were present at amino acid 149 of the yeast farnesyltransferase beta-subunit and the C-terminal amino acid of the CaaX protein substrate, but not when like charges were present at these positions . This evidence for electrostatic interaction between amino acid 149 and the C-terminal amino acid of CaaX protein substrates leads to the prediction that the C-terminal amino acid of the protein substrate binds near amino acid 149 of the yeast farnesyltransferase beta-subunit.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10729 - 34
Xenopus Cdc6 confers sperm binding competence to oocytes without inducing their maturation; Tian J et al.; Amphibian eggs normally require meiotic maturation to be competent for fertilization . A necessary prerequisite for this event is sperm binding, and we show that under normal physiological conditions this property is acquired at, but not before, meiotic maturation . Immature oocytes do not bind sperm, but injection of total egg poly(A)+ mRNA into immature oocytes confers sperm binding in the absence of meiotic maturation . Using an expression cloning approach we have isolated a single cDNA from egg poly(A)+ mRNA that can induce sperm binding in immature oocytes . The cDNA was found to encode Xenopus Cdc6, a protein that previously has been shown to function in initiation of DNA replication and cell cycle control . This unanticipated finding provides evidence of a link between a regulator of the cell cycle and alterations in cell surface properties that affect gamete binding.

J Biochem (Tokyo), 1997 Aug, 122(2), 438 - 52
Genomic organization and alternative splicing of human PACE4 (SPC4), kexin-like processing endoprotease; Tsuji A et al.; PACE4 (paired basic amino acid cleaving enzyme) is a member of a family of the mammalian kexin-like proprotein convertases containing a subtilisin-like catalytic domain . Previously we reported seven isoform mRNAs of PACE4 that vary in size and 3'-coding sequence {A . Tsuji et al . (1994) Biochem . Biophys . Res . Commun . 200, 943-950; K . Mori et al . (1997) J . Biochem . 121, 941-948} . To determine the origin of these isoforms, the entire human PACE4 gene has been isolated as a set of overlapping genomic DNA fragments, and analyzed by restriction enzyme digestion and nucleotide sequence determination . The human PACE4 gene spans at least 250 kb and is distributed over 25 exons that range in size from 39 to 1,422 base pairs . Human PACE4 gene is the largest kexin-like proprotein convertase gene reported to date . The most striking feature of its genomic structure is the size of the introns and the number of exons, although the general organization of signal peptide, propeptide, and catalytic domains, which are conserved in this family, is very similar to that reported for other kexin-like protease genes . The structural analysis of PACE4 genomic DNA indicates that multiple PACE4 transcripts are produced as a consequence of alternative RNA splicing events, including exon skipping, and differences in the usage of the inner 5'-splicing donor and polyadenylation sites . A major transcriptional start site was detected 314 bp upstream from the ATG translational start site by primer extension analysis . Sequence analysis of the 5'-flanking region revealed that PACE4 gene lacks TATA and CCAAT boxes in the proximal upstream region of the start site, although potential binding sites for several transcription factors including SP1, AP1, AP2, PEA3, Ets-1, GHF (growth hormone factor)-1, CREB (cyclic AMP response element binding protein), and basic helix-loop-helix proteins, were present . An unusual sequence of six tandem repeats of a nonadecamer (GGCCTGGGGGTTCACCTGC) containing an E box is found in the 5'-flanking region . These results suggest that PACE4 is not a constitutive gene product and its expression is regulated by various transcription factors.

J Cell Biol, 1997 Oct 6, 139(1), 245 - 56
An endothelial storage granule for tissue-type plasminogen activator; Emeis JJ et al.; In previous studies we have shown that, after stimulation by a receptor ligand such as thrombin, tissue-type plasminogen activator (tPA) and von Willebrand factor (vWf) will be acutely released from human umbilical vein endothelial cells (HUVEC) . However, the mechanisms involved in the secretion of these two proteins differ in some respects, suggesting that the two proteins may be stored in different secretory granules . By density gradient centrifugation of rat lung homogenates, a particle was identified that contained nearly all tPA activity and antigen . This particle had an average density of 1.11-1.12 g/ml, both in Nycodenz density gradients and in sucrose density gradients . A similar density distribution of tPA was found for a rat endothelial cell line and for HUVEC . After thrombin stimulation of HUVEC to induce tPA secretion, the amount of tPA present in high-density fractions decreased, concomitant with the release of tPA into the culture medium and a shift in the density distribution of P-selectin . vWf, known to be stored in Weibel-Palade bodies, showed an identical distribution to tPA in Nycodenz gradients . In contrast, the distribution in sucrose gradients of vWf from both rat and human lung was very different from that of tPA, suggesting that tPA and vWf were not present in the same particle . Using double-immunofluorescence staining of HUVEC, tPA- and vWf-containing particles showed a different distribution by confocal microscopy . The distribution of tPA also differed from the distribution of tissue factor pathway inhibitor, endothelin-1, and caveolin . By immunoelectronmicroscopy, immunoreactive tPA could be demonstrated in small vesicles morphologically different from the larger Weibel-Palade bodies . It is concluded that tPA in endothelial cells is stored in a not-previously-described, small and dense (d = 1.11-1.12 g/ml) vesicle, which is different from a Weibel-Palade body.

J Cell Biol, 1997 Oct 6, 139(1), 23 - 36
SOI1 encodes a novel, conserved protein that promotes TGN-endosomal cycling of Kex2p and other membrane proteins by modulating the function of two TGN localization signals; Brickner JH et al.; Localization of yeast Kex2 protease to the TGN requires a signal (TLS1) in its cytosolic tail (C-tail) . Mutation of TLS1 results in rapid transit of Kex2p to the vacuole . Isolation of suppressors of the Tyr713Ala mutation in TLS1 previously identified three SOI genes . SOI1, cloned by complementation of a sporulation defect, encodes a novel, hydrophilic 3,144-residue protein with homologues in Caenorhabditis elegans, Drosophila melanogaster, and humans . Epitope-tagged Soi1p existed in a detergent-insensitive, sedimentable form . Deletion of SOI1 impaired TGN localization of wild-type Kex2p and a fusion protein containing the C-tail of Ste13p, and also caused missorting of carboxypeptidase Y and accelerated vacuolar degradation of the Vps10p sorting receptor . Deletion of SOI1 improved retention of Tyr713Ala Kex2p in the pro-alpha-factor processing compartment but, unlike the original soi1 alleles, did not increase the half-life of Tyr713Ala Kex2p . These results suggested that Soi1p functions at two steps in the cycling of Kex2p and other proteins between the TGN and prevacuolar compartment (PVC) . This hypothesis was confirmed in several ways . Soi1p was shown to be required for optimal function of TLS1 . Suppression of the Tyr713Ala mutation by mutation of SOI1 was shown to be caused by activation of a second signal (TLS2) in the Kex2p C-tail . TLS2 delayed exit of Kex2p from the TGN, whereas TLS1 did not affect this step . We propose that Soi1p promotes cycling of TGN membrane proteins between the TGN and PVC by antagonizing a TGN retention signal (TLS2) and facilitating the function of a retrieval signal (TLS1) that acts at the PVC.

J Clin Invest, 1997 Oct 1, 100(7), 1888 - 93
Therapy for persistent hyperinsulinemic hypoglycemia of infancy . Understanding the responsiveness of beta cells to diazoxide and somatostatin; Kane C et al.; The neonatal disorder persistent hyperinsulinemic hypoglycemia of infancy (PHHI) arises as the result of mutations in the subunits that form the ATP-sensitive potassium (KATP) channel in pancreatic beta cells, leading to insulin hypersecretion . Diazoxide (a specific KATP channel agonist in normal beta cells) and somatostatin (octreotide) are the mainstay of medical treatment for the condition . To investigate the mechanism of action of these agents in PHHI beta cells that lack KATP currents, we applied patch clamp techniques to insulin-secreting cells isolated from seven patients with PHHI . Five patients showed favorable responses to medical therapy, and two were refractory . Our data reveal, in drug-responsive patients, that a novel ion channel is modulated by diazoxide and somatostatin, leading to termination of the spontaneous electrical events that underlie insulin hypersecretion . The drug-resistant patients, both of whom carried a mutation in one of the genes that encode KATP channel subunits, also lacked this novel K+ channel . There were no effects of diazoxide and somatostatin on beta cell function in vitro . These findings elucidate for the first time the mechanisms of action of diazoxide and somatostatin in infants with PHHI in whom KATP channels are absent, and provide a rationale for development of new therapeutic opportunities by K+ channel manipulation in PHHI treatment.

J Biol Chem, 1997 Oct 3, 272(40), 25360 - 6
The coiled-coil region of the G protein beta subunit . Mutational analysis of Ggamma and effector interactions; Pellegrino S et al.; The beta and gamma subunits of the heterotrimeric G proteins remain tightly associated throughout the signaling cycle as the betagamma dimer interacts with Galpha, receptors, and effectors . A coiled-coil structure involving alpha-helical segments at the N termini of the beta and gamma subunits contributes to the dimerization interface and has been implicated in effector signaling in yeast . Scanning mutagenesis of the coiled-coil region of the mammalian beta1 subunit was performed to examine the effect of point mutations on betagamma assembly and effector signaling in COS cell cotransfection assays . In addition to the E10K mutation described previously, mutations A11E, L14E, and I18E in beta1 were found to block betagamma association, as evidenced by the failure of the Gbeta mutants to undergo cytosolic translocation with cotransfected nonisoprenylated Ggamma . Although none of 14 beta1 point mutations prevented the betagamma-dependent activation of the c-Jun N-terminal kinase (JNK) effector pathway, the D20K point mutation enhanced JNK but not phospholipase C-beta2 activation . These findings implicate the coiled-coil region of Gbeta in JNK signaling, provide further evidence that the structural features of the betagamma complex mediating effector regulation may differ among effectors, and identify single codons in the mammalian beta subunit where mutation might yield a phenotype of defective signal transduction.

J Biol Chem, 1997 Oct 3, 272(40), 25200 - 9
The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing; Heinemeyer W et al.; The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes . The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different alpha-type subunits and two inner rings made of 7 different beta-type subunits . In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform beta-subunits . Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis . By directed mutational analysis of 20 S proteasomal beta-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity . Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable . Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities . The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal beta-type subunits is exerted by active subunits and occurs in the fully assembled particle . This trans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.

J Biol Chem, 1997 Oct 3, 272(40), 24927 - 33
A unique role of the beta-2 thyroid hormone receptor isoform in negative regulation by thyroid hormone . Mapping of a novel amino-terminal domain important for ligand-independent activation; Langlois MF et al.; Negative regulation by thyroid hormone is mediated by nuclear thyroid hormone receptors (TRs) acting on thyroid hormone response elements (TREs) . We examine here the role of human TR-beta2, a TR isoform with central nervous system-restricted expression, in the regulation of target genes whose expression are decreased by triiodothyronine (T3) . Using transient transfection studies, we found that TR-beta2 achieved significantly greater ligand-independent activation on the thyrotropin-releasing hormone (TRH) and common glycoprotein alpha-subunit genes than either TR-beta1 or TR-alpha1 . A chimeric TR-beta isoform containing the TR-beta2 amino terminus linked to the TR-alpha1 DNA- and ligand-binding domains functioned like the TR-beta2 isoform on these promoters, confirming that the amino terminus of TR-beta2 was both necessary and sufficient to mediate this effect . By constructing deletion mutants of the TR-beta2 amino terminus, we demonstrate that amino acids 89-116 mediate this function . This domain, important in ligand-independent activation on negative TREs, is discrete from a previously described activation domain in the amino-terminal portion of TR-beta2 . We conclude that the central nervous system-restricted TR-beta2 isoform has a unique effect on negative regulation by T3 that can be mapped to amino acids 89-116 of the amino terminus of the human TR-beta2.

J Biol Chem, 1997 Oct 3, 272(40), 24819 - 24
Cloning and characterization of a novel Cdc42-associated tyrosine kinase, ACK-2, from bovine brain; Yang W et al.; Cdc42 plays an important role in intracellular signaling pathways that influence cell morphology and motility and stimulate DNA synthesis . In attempts to determine whether nonreceptor tyrosine kinases play a fundamental role in Cdc42 signaling, we have cloned and biochemically characterized a new Cdc42-associated tyrosine kinase (ACK) from bovine brain . This tyrosine kinase, named ACK-2, has a calculated molecular mass of 83 kDa and shares a number of primary structural domains with the 120-kDa ACK (ACK-1) . The main differences between the primary structures of ACK-2 and ACK-1 occur in the amino- and carboxyl-terminal regions . Like ACK-1, ACK-2 binds exclusively to activated (GTP-bound) Cdc42 and does not bind to its closest homologs, e.g . activated Rac . ACK-2 could not be activated by addition of glutathione S-transferase (GST)-Cdc42(Q61L), a GTPase-defective mutant, or by GTPgammaS-loaded GST-Cdc42 in in vitro kinase assays . However, ACK-2 was activated when cotransfected with wild type Cdc42 or Cdc42(Q61L) and stably associated with Cdc42(Q61L) in vivo, indicating that ACK-2 interacts with active Cdc42 in cells . Furthermore, the tyrosine kinase activity of ACK-2 was stimulated both by epidermal growth factor and bradykinin, suggesting that ACK-2 may play a role in the signaling actions of both receptor tyrosine kinases or heterotrimeric G-protein-coupled receptors.

J Biol Chem, 1997 Oct 3, 272(40), 24763 - 6
Binding of Ku and c-Abl at the kinase homology region of DNA-dependent protein kinase catalytic subunit; Jin S et al.; The DNA-dependent protein kinase (DNA-PK) controls the repair of double-stranded DNA breaks in mammalian cells . The protein kinase subunit of DNA-PK (DNA-PKcs) is targeted to DNA breaks by association with the Ku DNA-binding heterodimer . Here we show that a Ku association site is present at the carboxyl terminus of DNA-PKcs (amino acids 3002-3850) near the protein kinase domain . Correspondingly, the nuclear c-Abl tyrosine kinase that associates with DNA-PK also binds to the kinase homology domain . The c-Abl SH3 domain binds to amino acids 3414-3850 of DNA-PKcs . c-Abl phosphorylates C-terminal fragments of DNA-PKcs, particularly amino acids 3414-3850 . c-Abl phosphorylation of DNA-PKcs disassociates the DNA-PKcs.Ku complex . Thus, Ku and c-Abl provide opposing functions with regard to DNA-PK activity.

J Biol Chem, 1997 Oct 3, 272(40), 24759 - 62
Identification by site-directed mutagenesis of three arginines in uncoupling protein that are essential for nucleotide binding and inhibition; Modriansky M et al.; Primary regulation of uncoupling protein is mediated by purine nucleotides, which bind to the protein and allosterically inhibit fatty acid-induced proton transport . To gain increased understanding of nucleotide regulation, we evaluated the role of basic amino acid residues using site-directed mutagenesis . Mutant and wild-type proteins were expressed in yeast, purified, and reconstituted into liposomes . We studied nucleotide binding as well as inhibition of fatty acid-induced proton transport in wild-type and six mutant uncoupling proteins . None of the mutations interfered with proton transport . Two lysine mutants and a histidine mutant had no effect on nucleotide binding or inhibition . Arg83 and Arg182 mutants completely lost both the ability to bind nucleotides and nucleotide inhibition . Surprisingly, the Arg276 mutant exhibited normal nucleotide binding, but completely lost nucleotide inhibition . To account for this dissociation between binding and inhibition, we propose a three-stage binding-conformational change model of nucleotide regulation of uncoupling protein . We have now identified three nucleotides by site-directed mutagenesis that are essential for nucleotide interaction with uncoupling protein.

EMBO J, 1997 Oct 1, 16(19), 5880 - 93
Sequential activation of phoshatidylinositol 3-kinase and phospholipase C-gamma2 by the M-CSF receptor is necessary for differentiation signaling; Bourette RP et al.; Binding of macrophage colony stimulating factor (M-CSF) to its receptor (Fms) induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for SH2-containing signaling proteins that relay growth and development signals . To determine whether a distinct signaling pathway is responsible for the Fms differentiation signal versus the growth signal, we sought new molecules involved in Fms signaling by performing a two-hybrid screen in yeast using the autophosphorylated cytoplasmic domain of the wild-type Fms receptor as bait . Clones containing SH2 domains of phospholipase C-gamma2 (PLC-gamma2) were frequently isolated and shown to interact with phosphorylated Tyr721 of the Fms receptor, which is also the binding site of the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) . At variance with previous reports, M-CSF induced rapid and transient tyrosine phosphorylation of PLC-gamma2 in myeloid FDC-P1 cells and this activation required the activity of the PI3-kinase pathway . The Fms Y721F mutation strongly decreased this activation . Moreover, the Fms Y807F mutation decreased both binding and phosphorylation of PLC-gamma2 but not that of p85 . Since the Fms Y807F mutation abrogates the differentiation signal when expressed in FDC-P1 cells and since this phenotype could be reproduced by a specific inhibitor of PLC-gamma, we propose that a balance between the activities of PLC-gamma2 and PI3-kinase in response to M-CSF is required for cell differentiation.

EMBO J, 1997 Sep 15, 16(18), 5730 - 41
Dual role of the C34 subunit of RNA polymerase III in transcription initiation; Brun I et al.; The C34 subunit of yeast RNA polymerase (pol) III is part of a subcomplex of three subunits which have no counterpart in the other two nuclear RNA polymerases . This subunit interacts with TFIIIB70 and is therefore thought to participate in pol III recruitment . To study the role of C34 in transcription, we have mutagenized RPC34, the gene encoding C34, and found that mutations affecting growth also altered C34 interaction with TFIIIB70 . The two mutant pol III that were purified had catalytic properties indistinguishable from those of the wild-type pol III on a poly{d(A-T)} template, while specific transcription of pol III genes in the presence of general transcription factors was impaired . The defect of the C34-1124 mutant enzyme could be compensated by increasing the amount of pol III present in the reaction, suggesting that the enzyme had a lower affinity for pre-initiation complexes . In contrast, the C34-1109 mutant enzyme was defective in transcription initiation due to impaired open complex formation . These observations demonstrate that the C34 subunit is a major determinant in pol III recruitment by the pre-initiation complex and further acts at a subsequent stage that involves the configuration of an initiation-competent form of RNA polymerase.

EMBO J, 1997 Sep 15, 16(18), 5550 - 61
A member of the Ste20/PAK family of protein kinases is involved in both arrest of Xenopus oocytes at G2/prophase of the first meiotic cell cycle and in prevention of apoptosis; Faure S et al.; We have identified new members (X-PAKs) of the Ste20/PAK family of protein kinases in Xenopus, and investigated their role in the process that maintains oocytes arrested in the cell cycle . Microinjection of a catalytically inactive mutant of X-PAK1 with a K/R substitution in the ATP binding site, also deleted of its Nter-half that contains the conserved domains responsible for binding of both Cdc42/Rac GTPases and SH3-containing proteins, greatly facilitates oocyte release from G2/prophase arrest by progesterone and insulin . Addition of the same X-PAK1 mutant to cell cycle extracts from unfertilized eggs induced apoptosis, as shown by activation of caspases and cytological changes in in vitro-assembled nuclei . This was suppressed by adding Bcl-2 or the DEVD peptide inhibitor of caspases, and rescued by competing the dominant-negative mutant with its constitutively active X-PAK1 counterpart . Such results indicate that X-PAK1 (or another member of the Xenopus Ste20/PAK family of protein kinases) is involved in arrest of oocytes at G2/prophase and prevention of apoptosis; thus death by apoptosis and release of healthy oocytes from cell cycle arrest may be linked . That cell cycle arrest protects oocytes from apoptosis is consistent with the finding that extracts from metaphase II-arrested oocytes are less sensitive to apoptotic signals than those from activated eggs.

EMBO J, 1997 Sep 15, 16(18), 5509 - 19
The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer; Johnson ES et al.; SMT3 is an essential Saccharomyces cerevisiae gene encoding a 11.5 kDa protein similar to the mammalian ubiquitin-like protein SUMO-1 . We have found that Smt3p, like SUMO-1 and ubiquitin, can be attached to other proteins post-translationally and have characterized the processes leading to the activation of the Smt3p C-terminus for conjugation . First, the SMT3 translation product is cleaved endoproteolytically to expose Gly98, the mature C-terminus . The presence of Gly98 is critical for Smt3p's abilities to be conjugated to protein substrates and to complement the lethality of a smt3Delta strain . Smt3p undergoes ATP-dependent activation by a novel heterodimeric enzyme consisting of Uba2p, a previously identified 71 kDa protein similar to the C-terminus of ubiquitin-activating enzymes (E1s), and Aos1p (activation of Smt3p), a 40 kDa protein similar to the N-terminus of E1s . Experiments with conditional uba2 mutants showed that Uba2p is required for Smt3p conjugation in vivo . Furthermore, UBA2 and AOS1 are both essential genes, providing additional evidence that they act in a distinct pathway whose role in cell viability is to conjugate Smt3p to other proteins.

EMBO J, 1997 Sep 1, 16(17), 5408 - 19
The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44; Dekker PJ et al.; Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70-Tim44 driving system . By blue native electrophoresis, we identify an approximately 90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence-containing preproteins . Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex . Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex . Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits . Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites . We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.

EMBO J, 1997 Sep 1, 16(17), 5289 - 98
DBF2, a cell cycle-regulated protein kinase, is physically and functionally associated with the CCR4 transcriptional regulatory complex; Liu HY et al.; CCR4, a general transcriptional regulator affecting the expression of a number of genes in yeast, forms a multi-subunit complex in vivo . Using the yeast two-hybrid screen, we have identified DBF2, a cell cycle-regulated protein kinase, as a CCR4-associated protein . DBF2 is required for cell cycle progression at the telophase to G1 cell cycle transition . DBF2 co-immunoprecipitated with CCR4 and CAF1/POP2, a CCR4-associated factor, and co-purified with the CCR4 complex . Moreover, a dbf2 disruption resulted in phenotypes and transcriptional defects similar to those observed in strains deficient for CCR4 or CAF1 . ccr4 and caf1 mutations, on the other hand, were found to affect cell cycle progression in a manner similar to that observed for dbf2 defects . These data indicate that DBF2 is involved in the control of gene expression and suggest that the CCR4 complex regulates transcription during the late mitotic part of the cell cycle.

J Virol, 1997 Oct, 71(10), 7393 - 403
Inhibition of parvovirus minute virus of mice replication by a peptide involved in the oligomerization of nonstructural protein NS1; Pujol A et al.; The large nonstructural protein NS1 of the minute virus of mice and other parvoviruses is involved in essential steps of the viral life cycle, such as DNA replication and transcriptional regulation, and is a major contributor to the toxic effect on host cells . Various biochemical functions, such as ATP binding, ATPase, site-specific DNA binding and nicking, and helicase activities, have been assigned to NS1 . Homo-oligomerization is a prerequisite for a number of proteins to be fully functional . In particular, helicases generally act as homo-oligomers . Indirect evidence of NS1 self-association has been recently obtained by a nuclear cotransport assay (J . P . Nuesch and P . Tattersall, Virology 196:637-651, 1993) . In order to demonstrate the oligomerizing property of NS1 in a direct way and localize the protein region(s) involved, the yeast two-hybrid system was used in combination with deletion mutagenesis across the whole NS1 molecule, followed by high-resolution mapping of the homo-oligomerization domain by a peptide enzyme-linked immunosorbent assay method . This study led to the identification of a distinct NS1 peptide that contains a bipartite domain involved in NS1 oligomerization . Furthermore, this isolated peptide was found to act as a specific competitive inhibitor and suppress NS1 helicase activity in vitro and parvovirus DNA replication in vivo, arguing for the involvement of NS1 oligomerization in these processes . Our results point to drug targeting of oligomerization motifs of viral regulatory proteins as a potentially useful antiviral strategy.

J Virol, 1997 Oct, 71(10), 7328 - 36
Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3; Kawaguchi Y et al.; The herpes simplex virus 1 (HSV-1) infected-cell protein 0 (ICP0) has the characteristics of a promiscuous transactivator of genes introduced into cells by infection or transfection . To identify cellular proteins interacting with ICP0, we used a domain of exon II of ICP0 that is known to be crucial for regulatory function of the protein as bait in the yeast two-hybrid screen . Our results were as follows . (i) A cDNA in a positive yeast colony was found to encode cyclin D3, a cell cycle regulator of G1 phase . (ii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to cyclin D3 specifically formed complexes with ICP0 contained in HSV-1-infected cell lysate . (iii) To enhance the expression of cyclin D3, the gene was inserted into the viral genome and overexpressed in infected cells . The overexpressed cyclin D3 colocalized with ICP0 in nuclear structures characteristic of ND10 and which earlier have been reported to contain ICP0 . (iv) The accumulation of cyclin D3 protein in Vero cells infected with an alpha0 deletion mutant was reduced relative to that of cells infected with wild-type virus or a recombinant virus in which the deleted alpha0 sequences were restored . (v) Lysates of Spodoptera frugiperda Sf9 cells doubly infected with baculoviruses genetically engineered to express cyclin D3 and cyclin-dependent kinase 4 (CDK4) phosphorylated GST fused to retinoblastoma protein (GST-pRb) but did not phosphorylate the GST-alpha0(20-241) or GST-alpha0(543-768) fusion protein or immunoprecipitated ICP0 proteins . Moreover, the chimeric GST-ICP0(exon II) protein shown to bind cyclin D3 had no effect on the activity of the kinase on GST-pRb when added to mixtures of lysates of Sf9 cells which coexpressed cyclin D3 and CDK4 . These results indicate that ICP0 interacts with, colocalizes with, and stabilizes the cyclin D3 cell cycle regulator and does not affect its interaction with the cyclin-dependent kinase.

Drug Metab Dispos, 1997 Sep, 25(9), 1059 - 64
Oxidation of methamphetamine and methylenedioxymethamphetamine by CYP2D6; Lin LY et al.; Methamphetamine (MeAmp) abuse has recently experienced a resurgence and approaches to the treatment of its addiction similar to those used with cocaine have been considered . As the treatment regimes are likely to use drugs whose metabolism is related to that of MeAmp, studies were initiated to establish the enzymology of the fate of MeAmp . This report describes investigations of the role of CYP2D6, the human isoform of the enzyme that catalyzes debrisoquine hydroxylation, in the 4-hydroxylation and N-demethylation of MeAmp . The results of studies with human liver microsomes including those from a genetically poor metabolizer with respect to CYP2D6, showing correlation between MeAmp and metoprolol hydroxylation and MDMA demethylenation, were consistent with a major involvement of CYP2D6 in the aromatic 4-hydroxylation of MeAmp . This was confirmed by studies with recombinant CYP2D6 expressed in yeast, which was also shown to effect the N-demethylation of MeAmp . The rate of the 4-hydroxylation reaction was substantially slower than the demethylenation of MDMA . In contrast to MeAmp, MDMA was not N-demethylated by CYP2D6 . Since CYP2D6 participates in the major steps of MeAmp metabolism, pharmacokinetic interactions are likely with other drug substrates proposed for the treatment of MeAmp addiction . Furthermore, the genetic polymorphism associated with the enzyme could manifest itself in abnormal responses to MeAmp.

Int Immunol, 1997 Sep, 9(9), 1329 - 38
Four P-like elements are required for optimal transcription of the mouse IL-4 gene: involvement of a distinct set of nuclear factor of activated T cells and activator protein-1 family proteins; Takemoto N et al.; We previously identified the P sequence as a critical regulatory element of the human IL-4 promoter . In the mouse IL-4 promoter, there are five elements homologous to the human P sequence designated conserved lymphokine element 0 (CLE0), P, P2, P3 and P4 . To characterize the role of these P-like elements and their binding factors in the native promoter, we did transient transfection and electrophoretic mobility shift assays (EMSA) . Transfection of EL-4 cells with the IL-4 promoter-reporter constructs carrying mutated P-like elements showed that four P-like elements, CLE0, P, P2 and P4, but not P3, were required for optimal activation of the IL-4 promoter . EMSA showed that both constitutive and inducible complexes bound to CLE0, P, P2 and P4, whereas only a constitutive complex bound to P3 . In competition and antibody supershift assays in EMSA, complexes formed with P or P2 proved to contain nuclear factor of activated T cells (NFAT) family proteins as major components . Activator protein (AP)-1 family proteins interacted with CLE0, P, P2 and P4 . NFAT/AP-1 complex formed only with P and P2 . Cross-competition assays among the P-like elements revealed element-specific and common complexes . Six tandem repeats of the P element linked to the SV40 promoter responded to phorbol 12-myristate 13-acetate, while that of other elements did not . It would thus appear that components of each P-like element-binding complexes are not identical and may coordinately contribute to transcriptional activity.

Development, 1997 Sep, 124(17), 3253 - 62
Targeted neuronal ablation: the role of pioneer neurons in guidance and fasciculation in the CNS of Drosophila; Hidalgo A et al.; Although pioneer neurons are the first to delineate the axon pathways, it is uncertain whether they have unique pathfinding abilities . As a first step in defining the role of pioneer neurons in the Drosophila embryonic CNS, we describe the temporal profile and trajectory of the axons of four pioneer neurons and show that they differ from previously published reports . We show, by targeted ablation of one, two, three or four pioneer neurons at a time, that (1) no single pioneer neuron is essential for axon tract formation, (2) the interaction between two pioneers is necessary for the establishment of each fascicle and (3) pioneer neurons function synergistically to establish the longitudinal axon tracts, to guide the fasciculation of follower neurons along specific fascicles and to prevent axons from crossing the midline.

Eur J Cell Biol, 1997 Sep, 74(1), 102 - 10
Histone acetyltransferases during the cell cycle and differentiation of Physarum polycephalum; Lusser A et al.; The dynamic state of histone acetylation is maintained by histone acetyltransferases (HATs) and deacetylases . Cellular fractionation of plasmodia of Physarum polycephalum and partial purification of subcellular fractions by chromatography revealed the existence of a cytoplasmic B-type and four nuclear A-type HATs . The cytoplasmic B-enzyme was highly specific for histone H4, causing di-acetylation of H4 in vitro . The nuclear enzymes (HAT-A1 to HAT-A4) accepted all core histones as substrates, but differed by the preference for certain histone species . Enzymes were analyzed during the naturally synchronous cell cycle of macroplasmodia . Each of the enzymes had its individual cell cycle activity pattern, indicating diverse functions in nuclear metabolism . When growing plasmodia were induced to undergo differentiation into dormant sclerotia, an additional enzyme (HAT-AS) appeared at a late stage of sclerotization which correlated with differentiation-specific histone synthesis and acetylation in the absence of DNA replication . When dormant sclerotia were induced to reenter the cell cycle, a further enzyme form (HAT-AG) appeared during a short time period prior to the first post-germination mitosis . This enzyme had a strong preference for H2B, correlating with the overproportional in vivo acetate incorporation in H2B . Both differentiation-associated HATs were undetectable in growing plasmodia . The results demonstrate that different functions of core histone acetylation are based on multiple enzyme forms that are independently regulated during the cell cycle . Transitions from one developmental stage into another are accompanied by specific enzyme forms . With respect to recent data in the literature it may be assumed that these HAT-forms are subunits of a HAT-complex whose composition changes during the cell cycle and differentiation.

Curr Genet, 1997 Jul, 32(1), 32 - 40
cDNA-mediated Ty recombination can take place in the absence of plus-strand cDNA synthesis, but not in the absence of the integrase protein; Nevo-Caspi Y et al.; Ty elements belong to the family of LTR-containing retrotransposons . Ty RNA is reverse transcribed by Ty-encoded proteins . The cDNA then transposes to new locations in the genome by a process that involves the integrase protein encoded by the element . We have previously shown that the Ty cDNA molecule can participate in recombination events with genomic Tys . In this study we have analyzed the role of the integrase protein in cDNA-mediated Ty recombination . We found that this process involves the integrase protein in a temperature-dependent manner . In addition we have investigated whether double-stranded DNA is the only molecule that can participate in cDNA-mediated Ty recombination . We have shown that mutations in the polypurine tract that abolish plus-strand synthesis do not prevent cDNA-mediated recombination, implying that other types of intermediates from the reverse-transcription process (e.g . single-stranded DNA or a hybrid RNA-cDNA molecule) can participate in cDNA-mediated Ty recombination.

Arch Biochem Biophys, 1997 Sep 15, 345(2), 271 - 7
Determination of FAD-binding domain in flavin-containing monooxygenase 1 (FMO1); Kubo A et al.; The flavin-containing monooxygenases (FMOs) are a family of flavoenzymes and contain one molecule of FAD per monomer . In order to demonstrate where FMO interacts with FAD, four mutants for the rat liver FMO1 protein were expressed in yeast and characterized . All four mutants were immunochemically similar to the unmodified form, although the contents of FAD in all four mutants were much lower than that in the unmodified form . Interestingly, the mutant generated by changing the first glycine of the proposed FAD-binding domain (GxGxxG) to alanine revealed catalytic activities, but was lower than those seen with the unmodified form . The conversion of the first glycine to alanine markedly increased and decreased the Km and Vmax values for imipramine N-oxidation, respectively . The other three mutants (RFMOm2, RFMOm3, and RFMOm4) were catalytically inactive . Our results suggest that three glycines, especially the second and third glycines, in the proposed FAD-binding domain were necessary for FMO to show catalytic activities . Using RFMOm1 and the unmodified form, the effects of n-octylamine on the activity of FMO1 were investigated . The activities of both wild-type and RFMOm1 enzymes for all of the compounds examined were enhanced by n-octylamine . The Km and Vmax values of both RFMOm1 and the unmodified form for imipramine N-oxidation were lowered and raised by n-octylamine, respectively.

Prog Nucleic Acid Res Mol Biol, 1998, 58, 263 - 99
Double-strand break-induced recombination in eukaryotes; Osman F et al.; Genetic recombination is of fundamental importance for a wide variety of biological processes in eukaryotic cells . One of the major questions in recombination relates to the mechanism by which the exchange of genetic information is initiated . In recent years, DNA double strand breaks (DSBs) have emerged as an important lesion that can initiate and stimulate meiotic and mitotic homologous recombination . In this review, we examine the models by which DSBs induce recombination, describe the types of recombination events that DSBs stimulate, and compare the genetic control of DSB-induced mitotic recombination in budding and fission yeasts.

Mol Biol Cell, 1997 Sep, 8(9), 1805 - 14
The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane; Cox JS et al.; The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments . Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself . In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response . We demonstrate that these two responses are intimately linked, forming different branches of the same pathway . Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

J Biol Chem, 1997 Sep 26, 272(39), 24563 - 71
Interaction of elongation factors TFIIS and elongin A with a human RNA polymerase II holoenzyme capable of promoter-specific initiation and responsive to transcriptional activators; Pan G et al.; Affinity chromatography on columns containing the immobilized monomeric transcriptional elongation factor TFIIS or the essential large subunit, Elongin A, of the trimeric elongation factor, Elongin, was used to purify a human RNA polymerase II holoenzyme from HeLa whole cell extract . This holoenzyme contained nearstoichiometric amounts of all the general transcription factors, TFIIB, TFIID (TBP + TAFIIs), TFIIE, TFIIF, and TFIIH, required to accurately initiate transcription in vitro at the adenovirus major late promoter . It behaved as a large complex, slightly smaller than 70 S ribosomes, during gel filtration chromatography, and contained nearly half the TFIID that was present in the extract used for the affinity chromatography . It also contained the cyclin-dependent kinase CDK8, a human homologue of the Saccharomyces cerevisiae holoenzyme subunit SRB10, and many other polypeptides . Efficient interaction of holoenzyme with TFIIS or Elongin A required only the amino-terminal region of either protein . These regions are similar in amino acid sequence but dispensable for TFIIS or Elongin to regulate elongation in vitro by highly purified RNA polymerase II . The transcriptional activators GAL4-VP16 and GAL4-Sp1 activated transcription in vitro by purified holoenzyme in the absence of any additional factors.

J Biol Chem, 1997 Sep 26, 272(39), 24363 - 70
p32 protein, a splicing factor 2-associated protein, is localized in mitochondrial matrix and is functionally important in maintaining oxidative phosphorylation; Muta T et al.; Human p32, originally cloned as a splicing factor 2-associated protein, has been reported to interact with a variety of molecules including human immunodeficiency virus Tat and complement 1q (C1q) . p32 protein is supposed to be in the nucleus and on the plasma membrane for the association with human immunodeficiency virus Tat and C1q, respectively . None of the interactions, however, is proven to have a physiological role . To investigate the physiological function of p32, we determined the intracellular localization of p32 . The fractionation of cells, fluorescent immunocytochemistry, and electron microscopic immunostaining show that p32 is exclusively localized in the mitochondrial matrix . We cloned a Saccharomyces cerevisiae homologue of human p32 gene, referred to yeast p30 gene . The yeast p30 protein is also localized in the mitochondrial matrix . The disruption of the p30 gene caused the growth retardation of yeast cells in a glycerol medium but not in a glucose medium, i.e . the impairment of the mitochondrial ATP synthesis . The growth impairment was restored by the introduction of the human p32 cDNA, indicating that p30 is a functional yeast counterpart of human p32 . Taken together, both p32 and p30 reside in mitochondrial matrix and play an important role in maintaining mitochondrial oxidative phosphorylation.

J Biol Chem, 1997 Sep 26, 272(39), 24105 - 8
Interaction of WW domains with hematopoietic transcription factor p45/NF-E2 and RNA polymerase II; Gavva NR et al.; NF-E2 is an erythroid-specific transcription factor required for expression of several erythroid-specific genes . By Far-Western blotting and yeast two-hybrid assay, we demonstrate that p45, the large subunit of NF-E2, is capable of binding to a specific set of WW domain-containing proteins, including the ubiquitin ligase hRPF1 . This binding is mediated through the interaction between the WW domains and a PY motif located within the amino-terminal region of p45 . Interestingly, the carboxyl-terminal domain of mammalian RNA polymerase II binds a similar set of WW domains to which p45 interacts with . We discuss the data in terms of possible new pathways through which the processes of transcriptional regulation by NF-E2 could be regulated in erythroid and megakaryote cells.

FEBS Lett, 1997 Sep 1, 414(1), 69 - 73
Cyclophilin active site mutants have native prolyl isomerase activity with a protein substrate; Scholz C et al.; The prolyl isomerase activity of cyclophilins is traditionally measured by an assay in which prolyl cis/trans isomerization in a chromogenic tetrapeptide is coupled with its isomer-specific cleavage by chymotrypsin . Two variants of mitochondrial cyclophilin with substitutions in the presumed active site (R73A and H144Q) are inactive in the protease-coupled assay, but show almost wild-type activity in an assay that is based on the catalysis of a proline-limited protein folding reaction . This prolyl isomerase assay is preferable, both because coupling with proteolysis is avoided and because an intact protein instead of a short peptide is used as a substrate . Possibly, some earlier conclusions about the catalytic mechanism and the involvement of the prolyl isomerase activity in the cellular function of immunophilins may need reevaluation.

EMBO J, 1997 Aug 15, 16(16), 5098 - 112
Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies; Yaneva M et al.; DNA-dependent protein kinase (DNA-PK or the scid factor) and Ku are critical for DNA end-joining in V(D)J recombination and in general non-homologous double-strand break repair . One model for the function of DNA-PK is that it forms a complex with Ku70/86, and this complex then binds to DNA ends, with Ku serving as the DNA-binding subunit . We find that DNA-PK can itself bind to linear DNA fragments ranging in size from 18 to 841 bp double-stranded (ds) DNA, as indicated by: (i) mobility shifts; (ii) crosslinking between the DNA and DNA-PK; and (iii) atomic-force microscopy . Binding of the 18 bp ds DNA to DNA-PK activates it for phosphorylation of protein targets, and this level of activation is not increased by addition of purified Ku70/86 . Ku can stimulate DNA-PK activity beyond this level only when the DNA fragments are long enough for the independent binding to the DNA of both DNA-PK and Ku . Atomic-force microscopy indicates that under such conditions, the DNA-PK binds at the DNA termini, and Ku70/86 assumes a position along the ds DNA that is adjacent to the DNA-PK.

EMBO J, 1997 Aug 15, 16(16), 5086 - 97
Two functionally distinct domains generated by in vivo cleavage of Nup145p: a novel biogenesis pathway for nucleoporins; Teixeira MT et al.; Nup145p is an essential yeast nucleoporin involved in nuclear export of polyadenylated RNAs . We demonstrate here that Nup145p is cleaved in vivo to yield two functionally distinct domains: a carboxy-terminal domain (C-Nup145p) which is located at the nuclear pore complex (NPC) and assembles into the Nup84p complex, and a GLFG-containing amino-terminal domain (N-Nup145p) which is not part of this complex . Whereas the essential C-Nup145p accomplishes the functions required for efficient mRNA export and normal NPC distribution, N-Nup145p, which is homologous to the GLFG-containing nucleoporins Nup100p and Nup116p, is not necessary for cell growth . However, the N-Nup145p becomes essential in a nup188 mutant background . Strikingly, generation of a free N-domain is a prerequisite for complementation of this peculiar synthetic lethal mutant . These data suggest that N- and C-domains of Nup145p perform independent functions, and that the in vivo cleavage observed is of functional importance.

EMBO J, 1997 Aug 15, 16(16), 5046 - 56
Nucleosome structure and positioning modulate nucleotide excision repair in the non-transcribed strand of an active gene; Wellinger RE et al.; Nucleotide excision repair (NER) is a major pathway to remove pyrimidine dimers (PDs), a class of DNA lesions generated by ultraviolet light . Since folding of DNA into nucleosomes restricts its accessibility and since transcription and DNA repair require access to DNA, nucleosome structure and positioning as well as the transcriptional state may affect DNA repair . We recently determined the chromatin structure of the yeast URA3 gene at high resolution and found multiple positions of nucleosomes as well as strand- and site-specific variation in DNA accessibility to DNase I (internal protected regions) . Here, the same high-resolution primer extension technique was used to investigate NER of PDs in the URA3 gene of a mini-chromosome in vivo . In the non-transcribed strand (NTS), fast repair correlates with PD locations in linker DNA and towards the 5' end of a positioned nucleosome . Slow repair correlates with the internal protected region of the nucleosome . This repair heterogeneity reflects a modulation of NER by positioned nucleosomes in the NTS . NER in the transcribed strand (TS) is fast, less heterogeneous and shows no correlation with chromatin structure . Apparently, transcription-coupled repair overrides chromatin modulation of NER in the TS . Heterogeneity in NER generated by chromatin structure on the NTS may contribute to heterogeneity in mutagenesis.

DNA Cell Biol, 1997 Aug, 16(8), 985 - 91
Identification of a new cellular protein that can interact specifically with DAN; Ozaki T et al.; Recently, we demonstrated that the DAN gene product contains a growth- and/or a tumor-suppressive activity in vitro . In the present work, using a yeast two-hybrid system, we searched for cellular proteins that can associate with the DAN gene product . A cDNA clone, termed DA41, was initially isolated from an adult rat lung cDNA library . The DA41 gene was expressed in all adult tissues examined, however, the levels of expression varied significantly among the different tissues . Like the DAN gene product, the DA41 protein was similarly restricted to the cytoplasm . Sequence analysis revealed that DA41 cDNA is 2,167 nucleotides in length and contains a single open reading frame (ORF) of 582 amino acids (61,945 daltons) . A homology search revealed that the DA41 gene product shares no structural similarity with those filed in the data base . In a synchronous 3Y1 cell culture, DA41 mRNA was expressed at a low level in quiescent cells; however, its level was significantly increased between the G1 and S phases of the cell cycle . On the other hand, the expression level of DAN mRNA did not change throughout the cell cycle progression . These results suggest that the DAN-DA41 complex might play a crucial role in the regulation of the cell cycle progression.

EMBO J, 1997 Aug 1, 16(15), 4770 - 6
A small nucleolar RNP protein is required for pseudouridylation of eukaryotic ribosomal RNAs; Bousquet-Antonelli C et al.; Eukaryotic rRNAs possess numerous post-transcriptionally modified nucleotides . The most abundant modifications, 2'-O-ribose methylation and pseudouridylation, occur in the nucleolus during rRNA processing . The nucleolus contains a large number of small nucleolar RNAs (snoRNAs) most of which can be classified into two distinct families defined by conserved sequence boxes and common associated proteins . The C and D box-containing snoRNAs are associated with fibrillarin, and most of them function as guide RNAs in site-specific ribose methylation of rRNAs . The nucleolar function of the other class of snoRNAs, which share box H and ACA elements and are associated with a glycine- and arginine-rich nucleolar protein, Gar1p, remains elusive . Here we demonstrate that the yeast Saccharomyces cerevisiae Gar1 snoRNP protein plays an essential and specific role in the overall pseudouridylation of yeast rRNAs . These results establish a novel function for Gar1 protein and indicate that the box H/ACA snoRNAs, or at least a subset of these snoRNAs, function in the site-specific pseudouridylation of rRNAs.

EMBO J, 1997 Aug 1, 16(15), 4746 - 59
Evidence that U5 snRNP recognizes the 3' splice site for catalytic step II in mammals; Chiara MD et al.; The first AG dinucleotide downstream from the branchpoint sequence (BPS) is chosen as the 3' splice site during catalytic step II of the splicing reaction . The mechanism and factors involved in selection of this AG are not known . Early in mammalian spliceosome assembly, U2AF65 binds to the pyrimidine tract between the BPS and AG . Here we show that U2AF65 crosslinking is replaced by crosslinking of three proteins of 110, 116 and 220 kDa prior to catalytic step II, and we provide evidence that all three proteins are components of U5 snRNP . These proteins interact with pre-mRNA in the region spanning from immediately downstream of U2 snRNP's binding site at the BPS to just beyond the 3' splice site . We also demonstrate that there are strict constraints on both the sequence and the distance between the BPS and AG for catalytic step II . Together, these observations suggest that U5 snRNP is positioned on the 3' splice site by an interaction (direct or indirect) with U2 snRNP bound at the BPS and by a direct interaction with the pyrimidine tract . The functional AG for catalytic step II may be specified, in turn, by its location with respect to the U5 snRNP binding site.

EMBO J, 1997 Aug 1, 16(15), 4639 - 49
Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system; Kluck RM et al.; In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria . We now describe in detail the crucial role of cytochrome c in this system . The mitochondrial fraction, when incubated with cytosol, releases cytochrome c . Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1 . CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes . Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis . Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required . The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redox-inactive . However, certain structural features of the molecule were required for this activity . Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors.

EMBO J, 1997 Aug 1, 16(15), 4560 - 7
Reconstitution of the protein insertion machinery of the mitochondrial inner membrane; Haucke V et al.; We have reconstituted the protein insertion machinery of the yeast mitochondrial inner membrane into proteoliposomes . The reconstituted proteoliposomes have a distinct morphology and protein composition and correctly insert the ADP/ATP carrier (AAC) and Tim23p, two multi-spanning integral proteins of the mitochondrial inner membrane . The reconstituted system requires a membrane potential, but not Tim44p or mhsp70, both of which are required for the ATP-driven translocation of proteins into the matrix . The protein insertion machinery can thus operate independently of the energy-transducing Tim44p-mhsp70 complex.

EMBO J, 1997 Aug 1, 16(15), 4540 - 8
Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation; Pilon M et al.; Degradation of misfolded secretory proteins has long been assumed to occur in the lumen of the endoplasmic reticulum (ER) . Recent evidence, however, suggests that such proteins are instead degraded by proteasomes in the cytosol, although it remains unclear how the proteins are transported out of the ER . Here we provide the first genetic evidence that Sec61p, the pore-forming subunit of the protein translocation channel in the ER membrane, is directly involved in the export of misfolded secretory proteins . We describe two novel mutants in yeast Sec61p that are cold-sensitive for import into the ER in both intact yeast cells and a cell-free system . Microsomes derived from these mutants are defective in exporting misfolded secretory proteins . These proteins become trapped in the ER and are associated with Sec61p . We conclude that misfolded secretory proteins are exported for degradation from the ER to the cytosol via channels formed by Sec61p.

EMBO J, 1997 Aug 1, 16(15), 4519 - 30
Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity; Sung TC et al.; Phospholipase D (PLD) genes are members of a superfamily that is defined by several highly conserved motifs . PLD in mammals has been proposed to play a role in membrane vesicular trafficking and signal transduction . Using site-directed mutagenesis, 25 point mutants have been made in human PLD1 (hPLD1) and characterized . We find that a motif (HxKxxxxD) and a serine/threonine conserved in all members of the PLD superfamily are critical for PLD biochemical activity, suggesting a possible catalytic mechanism . Functional analysis of catalytically inactive point mutants for yeast PLD demonstrates that the meiotic phenotype ensuing from PLD deficiency in yeast derives from a loss of enzymatic activity . Finally, mutation of an HxKxxxxD motif found in a vaccinia viral protein expressed in the Golgi complex results in loss of efficient vaccinia virus cell-to-cell spreading, implicating the viral protein as a member of the superfamily and suggesting that it encodes a lipid modifying or binding activity . The results suggest that vaccinia virus and hPLD1 may act through analogous mechanisms to effect viral cellular egress and vesicular trafficking, respectively.

Nat Struct Biol, 1997 Sep, 4(9), 751 - 9
Crystal structure of a PUT3-DNA complex reveals a novel mechanism for DNA recognition by a protein containing a Zn2Cys6 binuclear cluster; Swaminathan K et al.; PUT3 is a member of a family of at least 79 fungal transcription factors that contain a six-cysteine, two-zinc domain called a 'Zn2Cys6 binuclear cluster' . We have determined the crystal structure of the DNA binding region from the PUT3 protein bound to its cognate DNA target . The structure reveals that the PUT3 homodimer is bound asymmetrically to the DNA site . This asymmetry orients a beta-strand from one protein subunit into the minor groove of the DNA resulting in a partial amino acid-base pair intercalation and extensive direct and water-mediated protein interactions with the minor groove of the DNA . These interactions facilitate a sequence dependent kink at the centre of the DNA site and specify the intervening base pairs separating two DNA half-sites that are contacted in the DNA major groove . A comparison with the GAL4-DNA and PPR1-DNA complexes shows how a family of related DNA binding proteins can use a diverse set of mechanisms to discriminate between the base pairs separating conserved DNA half-sites.

Nat Struct Biol, 1997 Sep, 4(9), 744 - 50
Structure and mobility of the PUT3 dimer; Walters KJ et al.; The solution structure and backbone dynamics of the transcriptional activator PUT3 (31-100) has been characterized using NMR spectroscopy . PUT3 (31-100) contains three distinct domains: a cysteine zinc cluster, linker, and dimerization domain . The cysteine zinc cluster of PUT3 closely resembles the solution structure of GAL4, while the dimerization domain forms a long coiled-coil similar to that observed in the crystal structures of GAL4 and PPR1 . However, the residues at the N-terminal end of the coiled-coil behave very differently in each of these proteins . A comparison of the structural elements within this region provides a model for the DNA binding specificity of these proteins . Furthermore, we have characterized the dynamics of PUT3 to find that the zinc cluster and dimerization domains have very diverse dynamics in solution . The dimerization domain behaves as a large protein, while the peripheral cysteine zinc clusters have dynamic properties similar to small proteins.

J Immunol, 1997 Sep 15, 159(6), 2559 - 62
Regulation of Ku expression in normal murine B cells by stimuli that promote switch recombination; Zelazowski P et al.; DNA-dependent protein kinase, the catalytic subunit associated with the Ku heterodimer (Ku70/Ku86), has been implicated in switch recombination . Therefore, we tested whether certain stimuli known to promote switch recombination may act in part by inducing Ku expression . We find that resting B cells contain relatively low levels of nuclear Ku, but that Ku expression can be up-regulated by culturing the cells with two switch stimuli . First, IL-4 and CD40 engagement in combination, but neither of these stimuli acting alone, strongly induce Ku expression; Ku levels rise within 24 h, about 2 days before switch recombination is detected . Second, dextran-conjugated anti-IgD Abs strongly induce Ku expression, which is variably enhanced by IL-5, but not by IL-4 . Our data suggest that switch recombination may be regulated, at least in part, through changes in the nuclear expression of Ku.

Planta, 1997, 203 Suppl, S91 - 7
Light-regulated root gravitropism: a role for, and characterization of, a calcium/calmodulin-dependent protein kinase homolog; Lu YT et al.; Roots of many species grow downward (orthogravitropism) only when illuminated . Previous work suggests that this is a calcium-regulated response and that both calmodulin and calcium/calmodulin-dependent kinases participate in transducing gravity and light stimuli . A genomic sequence has been obtained for a calcium/calmodulin-dependent kinase homolog (MCK1) expressed in root caps, the site of perception for both light and gravity . This homolog consists of 7265 base pairs and contains 11 exons and 10 introns . Since MCK1 is expressed constitutively in both light and dark, it is unlikely that the light directly affects MCK1 expression, though the activity of the protein may be affected by light . In cultivars showing light-regulated gravitropism, we hypothesize that MCK1, or a homolog, functions in establishing the auxin asymmetry necessary for orthogravitropism.

FEMS Microbiol Rev, 1997 Aug, 21(1), 29 - 42
Expression cloning of fungal enzyme genes; a novel approach for efficient isolation of enzyme genes of industrial relevance; Dalboge H; Expression cloning is a relatively new method for fast and efficient cloning of enzyme genes from fungi that are known to make complex enzyme mixtures . In contrast to traditional cloning methods that are usually dependent on knowledge of at least a partial amino acid sequence in order to synthesize appropriate DNA probes or primers, the expression cloning method solely relies on access to reliable and sensitive enzyme assays . A representative expression cDNA library is made in Saccharomyces cerevisiae form the donor strain and relevant cDNA clones are detected directly based on the encoded enzyme activity . Thus, time-consuming enzyme purification and characterization steps are avoided . The method has been applied on the characterization of extracellular enzyme genes from the filamentous fungus Aspergillus aculeatus and has resulted in the isolation of 20 different enzyme genes such as endo-glucanases, xylanases, pectinases, proteases, hemicellulases and rhamnogalacturonan-degrading enzymes . All enzymes have been expressed in Aspergillus oryzae, purified and characterized . In the present review a description of the expression cloning technique will be given as well as examples of how the technique has been used in the exploration and characterization of a commercial enzyme product that is known to consist of a complex mixture of more than 25 different enzyme activities.

Virology, 1997 Sep 15, 236(1), 18 - 29
P32/TAP, a cellular protein that interacts with EBNA-1 of Epstein-Barr virus; Wang Y et al.; The Epstein-Barr virus (EBV) EBNA-1 protein has a central role in the maintenance of a latent EBV infection and is the only virus-encoded protein expressed in all EBV-associated tumors . EBNA-1 is required for replication of the episomal form of the latent viral genome and transactivates the latency C and LMP-1 promoters . The mechanisms by which EBNA-1 performs these functions are not known . Here we describe the cloning, expression, and characterization of a cellular protein, P32/TAP, which strongly interacts with EBNA-1 . We show that P32/TAP is expressed at high levels in Raji cells and is synthesized as a proprotein of 282 amino acids (aa) that is posttranslationally processed by a two-step cleavage process to yield a mature protein of 209 aa . It has been previously reported that P32/TAP is expressed on the cell surface . Our transient expression assays detected full-length P32/TAP (1-282 aa) in the cytoplasm while mature P32/TAP protein localized to the nucleus . Three lines of evidence support P32/TAP interaction with EBNA-1 . First, in the yeast two-hybrid system we mapped two interactive N-terminal regions of EBNA-1, aa 40-60 and aa 325-376, each of which contains arginine-glycine repeats . These regions interact with the C-terminal half of P32/TAP . Second, the full-length cytoplasmic P32/TAP protein can translocate nuclear EBNA-1 into the cytoplasm . Third, P32/TAP co-immunoprecipitated with EBNA-1 . We have confirmed that a Gal4 fusion protein containing the C-terminal region of P32/TAP (aa 244-282) transactivates expression from a reporter containing upstream Gal4-binding sites . Deletion of the P32/TAP interactive regions of EBNA-1 severely diminished EBNA-1 transactivation of FRTKCAT in transient expression assays . Our data suggest that interaction with P32/TAP may contribute to EBNA-1-mediated transactivation .

Biochem Biophys Res Commun, 1997 Sep 18, 238(2), 462 - 7
The highly basic ribosomal protein L41 interacts with the beta subunit of protein kinase CKII and stimulates phosphorylation of DNA topoisomerase IIalpha by CKII; Lee JH et al.; Protein kinase CKII (CKII) is a heterotetramer composed of two catalytic (alpha or alpha') and two regulatory (beta) subunits . Using the yeast two-hybrid system, we have identified the highly basic, ribosomal protein L41 as a cellular protein capable of interacting with the beta subunit of CKII . We show, furthermore, using purified proteins, that L41 protein and CKIIbeta associate directly in vitro . L41 protein is not a substrate for CKII phosphorylation, and it does not stimulate CKII activity with either beta-casein or synthetic peptide substrate (RRREEETEEE) . However, L41 protein stimulates the phosphorylation of DNA topoisomerase IIalpha by CKII by 2.5 times . Additionally, L41 protein enhances the autophosphorylation of CKIIalpha . The data indicate that L41 protein associates with CKII and can modulate its activity toward a specific substrate or substrates . The direct interaction of CKIIbeta with ribosomal proteins also suggests that CKIIbeta itself or CKII holoenzyme may be involved in ribosome assembly or translational control .

Biochem Biophys Res Commun, 1997 Sep 18, 238(2), 425 - 9
Characterization of the Ras binding domain of the RalGDS-related protein, RLF; O'gara MJ et al.; The Ras binding domain (RBD) of Rlf, a member of the RalGDS family of proteins, was characterized . Using an ELISA-based technique, the relative binding affinity of Rlf for a variety of mutant Ras proteins was determined . Rlf had significantly different binding characteristics than the Raf-1 RBD . The minimal effective Ras binding domain was defined as residues 657-778 using N- and C-terminal deletions of Rlf . Using the PHD algorithm, the secondary structure of this domain was predicted to be similar to the ubiquitin superfold previously identified in the Raf-1 RBD . When the predicted secondary structure of the Rlf-RBD was aligned with the known secondary structure of the Raf-RBD, amino acids in Raf-1 essential for Ras binding were found to also be conserved in Rlf . Consistent with this observation, alanine substitution of one of these residues (K687) in Rlf significantly reduced affinity for Ras-GTP .

Biochem Biophys Res Commun, 1997 Aug 28, 237(3), 735 - 40
Adenosine deaminase is a specific partner for the Grb2 isoform Grb3-3; Ramos-Morales F et al.; Grb3-3 is an isoform of Grb2, thought to arise by alternative splicing, that lacks a functional SH2 domain but retains functional SH3 domains, which allow interaction with other proteins through binding to prolinerich sequences . Several evidences suggest that besides common partners for Grb2 and Grb3-3, specific targets could exist . In order to find specific partners for Grb3-3, we have screened a human cDNA library by the yeast two-hybrid system with Grb3-3 as a bait . We have identified adenosine deaminase, an enzyme involved in purine metabolism whose deficiency is associated with severe combined immunodeficiency, as a Grb3-3 binding protein that is not able to bind to Grb2 . This interaction has been confirmed in vitro with GST fusion proteins and in vivo by coimmunoprecipitation experiments in NIH3T3 cells stably transfected with Grb3-3 . The functional significance of this finding is discussed.

Mutat Res, 1997 Aug, 384(2), 135 - 44
Report on the meeting held at City University, London on 16 December 1996: recent progress in DNA repair, mutation and recombination; Strike P et al.; This one-day DNA repair meeting was held at City University, London for the fourth consecutive year (P . Strike, Recent advances in DNA repair, mutation and recombination-a report of the meeting of the British Photobiology Society, London, 20 December 1993, Mutation Res . 315 (1994) 75-84; P . Strike, Recent advances in DNA repair and recombination: a report of the DNA Repair Network held at City University, London on 19 December 1994, Mutation Res . 337 (1995) 61-71 ; N.J . Jones, P . Strike, Recent research in DNA repair, mutation and recombination: a report of the DNA Repair Network meeting held at City University, London on 18 December 1995, Mutation Res . 364 (1996) 13-23) . The programme consisted of 18 pre-offered talks, there being no specially invited speakers . The meeting was well attended with over 150 participants representing 23 universities, research institutes and hospitals the length and breadth of the United Kingdom . Overseas attendees represented laboratories from Japan, The Netherlands and Spain . The topics of the talks were varied and included mismatch repair, the repair of oxidative damage, transcription-coupled repair and various mutagen-hypersensitive human syndromes . Although wide-ranging, one recurring themes in many of the talks was that of homologous proteins and the correspondence of repair mechanisms in bacterial, fungal and mammalian systems.

Mol Biochem Parasitol, 1997 Oct, 89(1), 61 - 72
The dihydroxyacetonephosphate pathway for biosynthesis of ether lipids in Leishmania mexicana promastigotes; Heise N et al.; Biosynthetic studies using both {14C}- and {32P}-labelled substrates and a cell-free system to synthesise 1-O-alkyl moieties in glycerolipids, have shown that the three initial steps in ether-lipid biosynthesis in Leishmania mexicana promastigotes resemble those described for mammals and are associated with glycosomes . Purified glycosomes were able to sequentially synthesise the first intermediates of the ether-lipid biosynthetic pathway {acyl-dihydroxyacetonephosphate (DHAP), alkyl-DHAP and acyl/alkyl-glycerol-3-phosphate (G3P)} when incubated in the presence of radiolabelled DHAP, palmitoyl-CoA, hexadecanol and NADPH . However, when glycosomes were incubated under the same conditions in the presence of radiolabelled G3P, a rapid synthesis of acyl-G3P and phosphatidic acid was observed without any formation of alkyl-G3P, suggesting that the enzyme alkyl-synthase recognises only acyl-DHAP as substrate . Both the DHAP acyltransferase (DHAP-AT) and alkyl-DHAP synthase activities were located inside glycosomes whereas the alkyl/acyl-DHAP oxidoreductase activity was associated with the cytoplasmic face of the glycosomal membrane . The G3P acyltransferase (G3P-AT) and lyso-phosphatidic acid acyltransferase activities were not found inside glycosomes . The results suggest that the DHAP-AT and G3P-AT activities are catalysed by two distinct enzymes associated with different sub-cellular compartments.

Nature, 1997 Sep 11, 389(6647), 194 - 8
Steroid receptor coactivator-1 is a histone acetyltransferase; Spencer TE et al.; Steroid receptors and coactivator proteins are thought to stimulate gene expression by facilitating the assembly of basal transcription factors into a stable preinitiation complex . What is not clear, however, is how these transcription factors gain access to transcriptionally repressed chromatin to modulate the transactivation of specific gene networks in vivo . The available evidence indicates that acetylation of chromatin in vivo is coupled to transcription and that specific histone acetyltransferases (HATs) target histones bound to DNA and overcome the inhibitory effect of chromatin on gene expression . The steroid-receptor coactivator SRC-1 is a coactivator for many members of the steroid-hormone receptor superfamily of ligand-inducible transcription factors . Here we show that SRC-1 possesses intrinsic histone acetyltransferase activity and that it also interacts with another HAT, p300/CBP-associated factor (PCAF) . The HAT activity of SRC-1 maps to its carboxy-terminal region and is primarily specific for histones H3 and H4 . Acetylation by SRC-1 and PCAF of histones bound at specific promoters may result from ligand binding to steroid receptors and could be a mechanism by which the activation functions of steroid receptors and associated coactivators enhance formation of a stable preinitiation complex, thereby increasing transcription of specific genes from transcriptionally repressed chromatin templates.

J Biol Chem, 1997 Sep 19, 272(38), 24072 - 80
The unique domain as the site on Lyn kinase for its constitutive association with the high affinity receptor for IgE; Vonakis BM et al.; Aggregation of the high affinity receptor for IgE (FcepsilonRI) leads to the phosphorylation of tyrosines on the beta and gamma chains of the receptor by the Src family kinase Lyn . We have studied the interaction between Lyn and the FcepsilonRI in vivo using a transfection-based approach . FcepsilonRI were stably transfected into Chinese hamster ovary cells . The small amount of endogenous Src family kinase was sufficient to phosphorylate receptor tyrosines upon extensive aggregation of FcepsilonRI but not after addition of dimers of IgE . Upon stable co-transfection of Lyn kinase into the cells, dimers were now able to stimulate receptor phosphorylation and the response to more extensive aggregation was enhanced . In contrast, co-transfection with catalytically inactive Lyn inhibited the aggregation-induced phosphorylation by the endogenous kinase, and a quantitatively similar inhibition was observed in cells transfected with the SH4-containing unique domain of Lyn . Consistent with the results of others using alternative approaches, our additional studies using a yeast two-hybrid system detected a direct interaction between intact Lyn or its unique domain and the C-terminal cytoplasmic domain of the beta chain but not with the receptor's other cytoplasmic domains.

J Biol Chem, 1997 Sep 19, 272(38), 23824 - 32
Identification and characterization of the AF-1 transactivation domain of thyroid hormone receptor beta1; Wilkinson JR et al.; Physiological responses to thyroid hormones are regulated by a set of nuclear receptors (TRs) related to the steroid receptor superfamily of ligand-dependent transcription factors . Although TR isoforms are highly conserved in their DNA binding, ligand binding, and carboxyl-terminal transactivation domains, their amino-terminal regions are completely divergent . We examined the contribution of these amino-terminal sequences to TRbeta1 function . An amino-terminally truncated version of rat TRbeta1 lacking amino acids 4-89 was impaired in hormone-dependent activation in both yeast and mammalian cells . This defect was not due to impairment of DNA binding, because the truncated receptor displayed enhanced homodimer binding on several different TREs, indicating that residues in the amino-terminal domain of TRbeta1 interfere with homodimerization of the receptor . The presence of an autonomous transactivation domain in the amino-terminal region was demonstrated by its ability to activate transcription in a constitutive manner when fused to the GAL4 DNA binding domain . Deletional analyses localized the residues comprising the amino-terminal transactivation region of TRbeta1 to 19 amino acids residing between residues 69 and 89 . Thus, the amino-terminal region of TRbeta1 contains an activation domain (AF-1) that can modulate the function of the receptor and may allow for the fine-tuning of receptor activity in various target tissues.

J Biol Chem, 1997 Sep 19, 272(38), 23469 - 72
The copper chaperone for superoxide dismutase; Culotta VC et al.; Copper is distributed to distinct localizations in the cell through diverse pathways . We demonstrate here that the delivery of copper to copper/zinc superoxide dismutase (SOD1) is mediated through a soluble factor identified as Saccharomyces cerevisiae LYS7 and human CCS (copper chaperone for SOD) . This factor is specific for SOD1 and does not deliver copper to proteins in the mitochondria, nucleus, or secretory pathway . Yeast cells containing a lys7Delta null mutation have normal levels of SOD1 protein, but fail to incorporate copper into SOD1, which is therefore devoid of superoxide scavenging activity . LYS7 and CCS specifically restore the biosynthesis of holoSOD1 in vivo . Elucidation of the CCS copper delivery pathway may permit development of novel therapeutic approaches to human diseases that involve SOD1, including amyotrophic lateral sclerosis.

Mol Hum Reprod, 1997 Aug, 3(8), 646 - 50
Sperm-zona interaction and recombinant DNA technology; Chapman NR et al.; Recombinant DNA technology has revolutionized our understanding of many biological systems . However, such techniques and their application have not been fully exploited in the study of sperm zona interaction . Using examples from other biological systems, we ourline several experimental approaches that are likely to significantly enhance our understanding of the gamete recognition process.

J Exp Med, 1997 Sep 15, 186(6), 921 - 9
Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination In vivo; Ouyang H et al.; Ku is a complex of two proteins, Ku70 and Ku80, and functions as a heterodimer to bind DNA double-strand breaks (DSB) and activate DNA-dependent protein kinase . The role of the Ku70 subunit in DNA DSB repair, hypersensitivity to ionizing radiation, and V(D)J recombination was examined in mice that lack Ku70 (Ku70(-/-)) . Like Ku80(-/-) mice, Ku70(-/-) mice showed a profound deficiency in DNA DSB repair and were proportional dwarfs . Surprisingly, in contrast to Ku80(-/-) mice in which both T and B lymphocyte development were arrested at an early stage, lack of Ku70 was compatible with T cell receptor gene recombination and the development of mature CD4+CD8- and CD4-CD8+ T cells . Our data shows, for the first time, that Ku70 plays an essential role in DNA DSB repair, but is not required for TCR V(D)J recombination . These results suggest that distinct but overlapping repair pathways may mediate DNA DSB repair and V(D)J recombination.

J Gen Virol, 1997 Sep, 78 ( Pt 9), 2259 - 67
Augmentation of human T cell leukaemia virus type I Tax transactivation by octamer binding sites; Marriott SJ et al.; The human T cell leukaemia virus type I (HTLV-1) Tax protein is an activator of viral and cellular gene expression . Tax does not bind DNA directly, but does interact with cellular DNA binding proteins . These interactions bring Tax to a specific group of promoters and may help to determine the specificity of Tax transactivation . Previous studies have demonstrated that the activity of Tax, when tethered to a given promoter, is enhanced by the presence of adjacent transcription factor binding sites . To examine the specificity of this augmentation, a series of transcription factor binding sites was tested for the ability to enhance the activity of a Gal-Tax fusion protein . The greatest increase in Gal-Tax activity was observed when an octamer binding site was placed adjacent to the Gal4 binding sites . However, the octamer binding site failed to independently function as a Tax responsive element in the absence of an adjacent Tax-tethering element . Oct-2 was not required for augmentation of Gal-Tax activity as this enhancement was observed in BHK-21 cells, which lack Oct-2 . The ability of octamer binding sites to augment transcription was specific for Gal-Tax, as compared to other transactivators . Taken together, these results demonstrate that the degree of Tax transactivation can be influenced by the elemental composition of the target promoter.

Mol Biol Rep, 1997 Aug, 24(3), 197 - 207
Nuclear matrix, dynamic histone acetylation and transcriptionally active chromatin; Davie JR; The nuclear matrix, the RNA-protein skeleton of the nucleus, has a role in the organization and function of nuclear DNA . Nuclear processes associated with the nuclear matrix include transcription, replication and dynamic histone acetylation . Nuclear matrix proteins, which are tissue and cell type specific, are altered with transformation and state of differentiation . Transcription factors are associated with the nuclear matrix, with the spectra of nuclear matrix bound factors being cell type specific . There is compelling evidence that the transcription machinery is anchored to the nuclear matrix, and the chromatin fiber is spooled through this complex . Transcriptionally active chromatin domains are associated with dynamically acetylated histones . The energy exhaustive process of dynamic histone acetylation has several functions . Acetylation of the N-terminal tails of the core histones alters nucleosome and higher order chromatin structure, aiding transcriptional elongation and facilitating the binding of transcription factors to nucleosomes associated with regulatory DNA sequences . Histone acetylation can manipulate the interactions of regulatory proteins that bind to the N-terminal tails of the core histones . Lastly, dynamic acetylation may contribute to the transient attachment of transcriptionally active chromatin to the nuclear matrix . Reversible histone acetylation is catalyzed by histone acetyltransferase and deacetylase, enzymes associated with the nuclear matrix . The recent isolation and characterization of histone acetyltransferase and deacetylase reveals that these enzymes are related to transcriptional regulators, providing us with new insights about how these enzymes are targeted to nuclear matrix sites engaged in transcription.

Plant Mol Biol, 1997 Aug, 34(6), 891 - 6
Characterization of Zea mays endosperm C-24 sterol methyltransferase: one of two types of sterol methyltransferase in higher plants; Grebenok RJ et al.; We report the characterization of a higher-plant C-24 sterol methyltransferase by yeast complementation . A Zea mays endosperm expressed sequence tag (EST) was identified which, upon complete sequencing, showed 46% identity to the yeast C-24 methyltransferase gene (ERG6) and 75% and 37% amino acid identity to recently isolated higher-plant sterol methyltransferases from soybean and Arabidopsis, respectively . When placed under GALA regulation, the Z . mays cDNA functionally complemented the erg6 mutation, restoring ergosterol production and conferring resistance to cycloheximide . Complementation was both plasmid-dependent and galactose-inducible . The Z . mays cDNA clone contains an open reading frame encoding a 40 kDa protein containing motifs common to a large number of S-adenosyl-L-methionine methyltransferases (SMTs) . Sequence comparisons and functional studies of the maize, soybean and Arabidopsis cDNAs indicates two types of C-24 SMTs exist in higher plants.

Plant Mol Biol, 1997 Aug, 34(6), 867 - 77
Cloning, molecular characterization and expression pattern of a strawberry ripening-specific cDNA with sequence homology to pectate lyase from higher plants; Medina-Escobar N et al.; A strawberry fruit cDNA showing sequence similarity to higher-plant pectate lyase genes has been isolated by differential screening of a strawberry fruit cDNA subtractive library . The cDNA contains a 396 amino acids open reading frame corresponding to a 44.8 kDa protein . The transcript is predominantly expressed in ripe fruits and was not detected at high levels in any other plant tissues . The removal of the achenes from unripe green fruits induced the expression of this putative pectate lyase gene . In common with other ripening related genes in strawberry, this induction was partially inhibited by treatment of de-achened fruit with the auxin NAA . Southern blot analysis of genomic DNA indicates that in strawberry there is more than one putative pectate lyase gene . We propose that the ripe fruit expression of this strawberry gene with similarity to pectate lyases could be related to cell wall pectin degradation contributing to strawberry fruit softening.

Fungal Genet Biol, 1997 Jun, 21(3), 292 - 301
Cloning heterologous genes: problems and approaches; Agnan J et al.; Model fungi such as Neurospora crassa, Aspergillus niger, and Saccharomyces cerevisiae have provided a wealth of genetic information and are currently the object of cooperative genome sequencing projects . Many agriculturally and medically economic important pathogenic fungi, however, are less well characterized, which makes it difficult to study their genes and gene products . Gene sequences from model fungi offer a unique opportunity to clone cognate genes from pathogenic counterparts . In this review, we propose three basic strategies for cloning such genes: Functional complementation, sequence similarity, and genetic linkage . These strategies involve Southern hybridization, cloning, library screening, genetic complementation, and the polymerase chain reaction . We review the major problems encountered using these strategies and outline useful solutions to these difficulties.

Yeast, 1997 Sep 15, 13(11), 1059 - 64
The effect of a suppressed rad52 mutation on the suppression of rad6 by srs2; Nguyen MM et al.; We have cloned a suppressor of a temperature-sensitive rad52 allele and found it to be a mutation in PUP1, a gene encoding a protease subunit of the 20s proteasome . This identity prompted us to examine the interrelationship among PUP1, RAD52, SRS2 and RAD6 because srs2 mutations not only suppress some rad52 mutations but also suppress deletions of rad6, a gene encoding a protein in the ubiquination-dependent proteolysis pathway . We have found that while srs2 suppresses the UV sensitivity of rad6 in the presence of RAD52, srs2 cannot suppress rad6 when the temperature-sensitive allele of rad52 is present . This inability of srs2 to suppress rad6 is irrespective of the incubation temperature or whether pup1 is suppressing the temperature-sensitive rad52 mutation.

J Neurosci, 1997 Sep 15, 17(18), 6947 - 51
Protein phosphorylation and taurine biosynthesis in vivo and in vitro; Tang XW et al.; Taurine is known to be involved in many important physiological functions . Here we report that both in vivo and in vitro the taurine-synthesizing enzyme in the brain, namely cysteine sulfinic acid decarboxylase (CSAD), is activated when phosphorylated and inhibited when dephosphorylated . Furthermore, protein kinase C and protein phosphatase 2C have been identified as the enzymes responsible for phosphorylation and dephosphorylation of CSAD, respectively . In addition, the effect of neuronal depolarization on CSAD activity and 32P incorporation into CSAD in neuronal cultures is also included . A model to link neuronal excitation and CSAD activation by a Ca2+-dependent protein kinase is proposed.

Nucleic Acids Res, 1997 Sep 15, 25(18), 3594 - 604
A computational genomics approach to the identification of gene networks; Wagner A; To delineate the astronomical number of possible interactions of all genes in a genome is a task for which conventional experimental techniques are ill-suited . Sorely needed are rapid and inexpensive methods that identify candidates for interacting genes, candidates that can be further investigated by experiment . Such a method is introduced here for an important class of gene interactions, i.e., transcriptional regulation via transcription factors (TFs) that bind to specific enhancer or silencer sites . The method addresses the question: which of the genes in a genome are likely to be regulated by one or more TFs with known DNA binding specificity? It takes advantage of the fact that many TFs show cooperativity in transcriptional activation which manifests itself in closely spaced TF binding sites . Such 'clusters' of binding sites are very unlikely to occur by chance alone, as opposed to individual sites, which are often abundant in the genome . Here, statistical information about binding site clusters in the genome, is complemented by information about (i) known biochemical functions of the TF, (ii) the structure of its binding site, and (iii) function of the genes near the cluster, to identify genes likely to be regulated by a given transcription factor . Several applications are illustrated with the genome of Saccharomyces cerevisiae , and four different DNA binding activities, SBF, MBF, a sub-class of bHLH proteins and NBF . The technique may aid in the discovery of interactions between genes of known function, and the assignment of biological functions to putative open reading frames.

J Biol Chem, 1997 Sep 12, 272(37), 23398 - 406
The basic helix-loop-helix-zipper transcription factor USF1 regulates expression of the surfactant protein-A gene; Gao E et al.; Expression of the rabbit pulmonary surfactant protein A (SP-A) gene is lung-specific, occurs primarily in type II cells, and is developmentally regulated . We previously identified two E-box-like enhancers, termed the distal binding element (DBE) and proximal binding element (PBE), in the 5'-flanking region of the rabbit SP-A gene . In the present study, the PBE was used to screen a rabbit fetal lung cDNA expression library; a cDNA insert was isolated which is highly similar in sequence to human upstream stimulatory factor 1 (hUSF1) . By use of reverse transcription polymerase chain reaction, two isoforms of rabbit USF1 (rUSF1) mRNAs were identified in fetal rabbit lung and other tissues . The levels of rUSF1 mRNAs reach a peak in fetal rabbit lung at 23 days gestation, in concert with the time of initiation of SP-A gene transcription . Binding complexes of nuclear proteins obtained from fetal rabbit lung tissue and isolated type II cells with the DBE and PBE were supershifted by the addition of anti-rUSF1 IgG . Binding activity was enriched in type II cells compared with lung fibroblasts . Overexpression of rUSF1s in A549 adenocarcinoma cells positively regulated SP-A promoter activity of cotransfected reporter gene constructs . It is suggested that rUSF1s, which bind to two E-box elements in the SP-A gene 5'-flanking region, may serve a key role in the regulation of SP-A gene expression in pulmonary type II cells.

Cell Tissue Res, 1997 Oct, 290(1), 1 - 10
GAL4-responsive UAS-tau as a tool for studying the anatomy and development of the Drosophila central nervous system; Ito K et al.; To improve the quality of cytoplasmic labelling of GAL4-expressing cells in Drosophila enhancer-trap and transgenic strains, a new GAL4-responsive reporter UAS-tau, which features a bovine tau cDNA under control of a yeast upstream activation sequence (UAS), was tested . Tau, a microtubule-associated protein, is distributed actively and evenly into all cellular processes . Monoclonal anti-bovine Tau antibody reveals the axonal structure of the labelled cells with detail similar to that of Golgi impregnation . We demonstrate that the UAS-tau system is especially useful for studying processes of differentiation and reorganisation of identified neurones during postembryonic development.

Biochemistry, 1997 Sep 9, 36(36), 10930 - 5
Deletion of amino acids 261-269 in the brown fat uncoupling protein converts the carrier into a pore; Gonzalez-Barroso MM et al.; The uncoupling protein (UCP) from brown adipose tissue mitochondria is a carrier that catalyzes proton re-entry into the matrix and thus dissipates the proton electrochemical potential gradient as heat . UCP activity is regulated: purine nucleotides inhibit while fatty acids activate transport . We have previously reported that sequence 261-269 of the UCP has a closely related counterpart in the adenine nucleotide translocator, as well as in the DNA binding domain of the estrogen receptor . Site-directed mutagenesis of the UCP showed that deletion of amino acids 267-269 in the UCP abolished nucleotide inhibition {Bouillaud, F., et al . (1994) EMBO J . 13, 1990-1997} . Complete deletion of the homologous domain (UCPDelta9) produced a highly deleterious mutant that collapsed the mitochondrial membrane potential and halted yeast growth . Since under our growth conditions revertants appeared rapidly, it was not possible to characterize this mutant . In this article, we have designed conditions to isolate mitochondria containing significant amounts of the UCPDelta9 mutant protein . These mitochondria show no respiratory control and are insensitive to nucleotides . Investigation of the permeability properties revealed that UCPDelta9 mitochondria swell rapidly in potassium salts in the absence of valinomycin, thus indicating a loss of specificity . The size exclusion properties of this mutant were determined with polyethylene glycols of various molecular masses (400-20000 Da), and it was found that UCPDelta9 can catalyze permeation of molecules of up to 1000 Da . We conclude that the deletion of amino acids 261-269 converts the UCP into an unspecific pore.

J Cell Biol, 1997 Sep 8, 138(5), 975 - 85
Structure of L-A virus: a specialized compartment for the transcription and replication of double-stranded RNA; Caston JR et al.; The genomes of double-stranded (ds)RNA viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment-the viral capsid . We have used cryoelectron microscopy and three-dimensional image reconstruction to study this compartment in the case of L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD) . At 16-A resolution, we distinguish multiple domains in the elongated Gag subunits, whose nonequivalent packing is reflected in subtly different morphologies of the two protomers . Small holes, 10-15 A across, perforate the capsid wall, which functions as a molecular sieve, allowing the exit of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes . Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is an A-form duplex, and that RNA filaments emanating from disrupted virions often consist of two or more closely associated duplexes . Nuclease protection experiments confirm that the genome is entirely sequestered inside full capsids, but it is packed relatively loosely; in L-A, the center-to-center spacing between duplexes is 40-45 A, compared with 25-30 A in other double-stranded viruses . The looser packing of L-A RNA allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the polymerase immobilized on the inner capsid wall.

J Biol Chem, 1997 Sep 5, 272(36), 22679 - 84
LOK is a novel mouse STE20-like protein kinase that is expressed predominantly in lymphocytes; Kuramochi S et al.; We have identified a new gene, designated lok (lymphocyte-oriented kinase), that encodes a 966-amino acid protein kinase whose catalytic domain at the N terminus shows homology to that of the STE20 family members involved in mitogen-activated protein (MAP) kinase cascades . The non-catalytic domain of LOK does not have any similarity to that of other known members of the family . There is a proline-rich motif with Src homology region 3 binding potential, followed by a long coiled-coil structure at the C terminus . LOK is expressed as a 130-kDa protein, which was detected predominantly in lymphoid organs such as spleen, thymus, and bone marrow, in contrast to other mammalian members of the STE20 family . LOK phosphorylated itself as well as substrates such as myelin basic protein and histone IIA on serine and threonine residues but not on tyrosine residues, establishing LOK as a novel serine/threonine kinase . When coexpressed in COS7 cells with the known MAP kinase isoforms (ERK, JNK, and p38), LOK activated none of them in contrast to PAK- and GCK-related kinases . These results suggest that LOK could be involved in a novel signaling pathway in lymphocytes, which is distinct from the known MAP kinase cascades.

J Biol Chem, 1997 Sep 5, 272(36), 22526 - 30
Nm23/PuF does not directly stimulate transcription through the CT element in vivo; Michelotti EF et al.; Decreased levels of the nm23 gene product have been correlated with increased tumor metastatic potential in a variety of malignancies . At least a subset of the regulatory properties of Nm23 has been proposed to be due to transactivation of the human c-myc oncogene through binding to a homopyrimidine tract 140 base pairs upstream of the transcription start site (termed the CT element or the PuF site) . Conventional transcription factors possess DNA binding and transactivation domains; Nm23 fusion proteins were used to address two questions . First, if provided with a well characterized DNA binding domain, does Nm23 possess a transactivation domain capable of stimulating transcription of an appropriate reporter? Second, if provided with a potent transactivation domain, is the DNA binding of Nm23 of sufficient specificity and affinity to direct the fusion protein to a CT-dependent reporter? Since reporter gene expression was not stimulated in either case, we conclude that Nm23 does not directly stimulate transcription through binding to the CT element and that its antimetastatic and other reported functions are likely due to other biochemical activities.

J Biol Chem, 1997 Sep 5, 272(36), 22456 - 63
Improved activity and modulated action pattern obtained by random mutagenesis at the fourth beta-alpha loop involved in substrate binding to the catalytic (beta/alpha)8-barrel domain of barley alpha-amylase 1; Matsui I et al.; The functionality of the sequence Arg183-Gly184-Tyr185 of the substrate binding fourth beta-alpha loop in the (beta/alpha)8-barrel of barley alpha-amylase isozyme 1 (AMY1) was studied by random mutagenesis . A motif of polar Gly184 hydrophobic residues was present in active mutants, selected by starch plate screening of yeast transformants . Gly184 was important, probably due to the carbonyl group binding to Ca2+ and the spatial proximity of Phe181 . Mutation of both flanking residues as in Ser183-Gly184-Met185 (SGM-) and TGL-AMY1 decreased the Ca2+ affinity . SGM-AMY1 has 2-fold increased activity for amylose but reduced activity on maltooligosaccharides, whereas KGY-AMY1 has up to 3-fold elevated activity toward the oligosaccharides . TGL-AMY1 has modest activity on all substrates . Shifted action pattern on maltooligosaccharides for NGY-, SGM-, and TGL-AMY1 support that Arg183 in wild type is located at subsites +1 and +2, accommodating two sugar rings toward the reducing end from the site of cleavage . In the crystal structure of barley alpha-amylase 2 (AMY2), Lys182 (equivalent to AMY1 Arg183) is hydrogen-bonded with sugar OH-3 in subsite +2 . Higher Ki app for acarbose inhibition of KGY-AMY1 and parent AMY1 compared with the other mutants suggests favorable substrate interactions for Arg/Lys183 . KGY-AMY1 was not inhibited by the AMY2-specific proteinaceous barley alpha-amylase/subtilisin inhibitor, although Lys182 of AMY2 is salt-linked to the inhibitor.

Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9979 - 83
Expression of an ortholog of replication protein A1 (RPA1) is induced by gibberellin in deepwater rice; van der Knaap E et al.; Internodes of deepwater rice are induced to grow rapidly when plants become submerged . This adaptation enables deepwater rice to keep part of its foliage above the rising flood waters during the monsoon season and to avoid drowning . This growth response is, ultimately, elicited by the plant hormone gibberellin (GA) . The primary target tissue for GA action is the intercalary meristem of the internode . Using differential display of mRNA, we have isolated a number of genes whose expression in the intercalary meristem is regulated by GA . The product of one of these genes was identified as an ortholog of replication protein A1 (RPA1) . RPA is a heterotrimeric protein involved in DNA replication, recombination, and repair and also in regulation of transcription . A chimeric construct, in which the single-stranded DNA-binding domain of rice RPA1 was spliced into the corresponding region of yeast RPA1, was able to complement a yeast rpa1 mutant . The transcript level of rice RPA1 is high in tissues containing dividing cells . RPA1 mRNA levels increase rapidly in the intercalary meristem during submergence and treatment with GA before the increase in the level of histone H3 mRNA, a marker for DNA replication.

Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9693 - 8
Rer1p as common machinery for the endoplasmic reticulum localization of membrane proteins; Sato K et al.; Rer1p, a Golgi membrane protein, is required for the correct localization of an endoplasmic reticulum (ER) membrane protein, Sec12p, by a retrieval mechanism from the cis-Golgi to the ER . To test whether or not the role of Rer1p is common to multiple ER membrane proteins, we examined the localization of two other ER membrane proteins, Sec71p and Sec63p, in the wild-type and rer1 mutant yeast cells, using their fusions with an alpha-mating factor precursor (Mfalpha1p) . Although Sec71p and Sec63p have completely different topology from Sec12p, their Mfalpha1p fusion proteins were also mislocalized to the trans-Golgi in the rer1 mutant . Overexpression of these fusions caused their mislocalization to the trans-Golgi even in the wild-type cells, and this mislocalization was partially suppressed by the co-overexpression of Rer1p . Either Sec71p or an artificial chimeric protein whose ER localization depends on Rer1p gave a competitive effect on the localization of the Mfalpha1-Sec71p fusion, which was abolished in rer1 . Thus, Rer1p appears to be one of the common limiting components in the retrieval machinery for ER membrane proteins . The results also suggest that Sec71p and Sec63p depend on ER-Golgi recycling, at least partly, for ER localization . We also examined the effect of a mutation in alpha-COP, a subunit of yeast coatomer, on the localization of these ER membrane proteins . The Mfalpha1p fusions of Sec12p, Sec71p, and Sec63p were all more or less mislocalized in ret1-1 . These observations imply that the roles of Rer1p and coatomer are much more general than thought before.

Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9573 - 8
Phylogeny of mRNA capping enzymes; Wang SP et al.; The m7GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by the sequential action of three enzymes: RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase . A multifunctional polypeptide containing all three active sites is encoded by vaccinia virus . In contrast, fungi and Chlorella virus encode monofunctional guanylyltransferase polypeptides that lack triphosphatase and methyltransferase activities . Transguanylylation is a two-stage reaction involving a covalent enzyme-GMP intermediate . The active site is composed of six protein motifs that are conserved in order and spacing among yeast and DNA virus capping enzymes . We performed a structure-function analysis of the six motifs by targeted mutagenesis of Ceg1, the Saccharomyces cerevisiae guanylyltransferase . Essential acidic, basic, and aromatic functional groups were identified . The structural basis for covalent catalysis was illuminated by comparing the mutational results with the crystal structure of the Chlorella virus capping enzyme . The results also allowed us to identify the capping enzyme of Caenorhabditis elegans . The 573-amino acid nematode protein consists of a C-terminal guanylyltransferase domain, which is homologous to Ceg1 and is strictly conserved with respect to all 16 amino acids that are essential for Ceg1 function, and an N-terminal phosphatase domain that bears no resemblance to the vaccinia triphosphatase domain but, instead, has strong similarity to the superfamily of protein phosphatases that act via a covalent phosphocysteine intermediate.

Nat Genet, 1997 Sep, 17(1), 100 - 3
The Werner syndrome protein is a DNA helicase; Gray MD et al.; Werner syndrome (WS) is an uncommon autosomal recessive disorder characterized by premature aging . The clinical manifestations of WS, including atherosclerosis and osteoporosis, appear early in adulthood, and death in the fourth to sixth decade commonly ensues from myocardial infarction or cancer . In accord with the aging phenotype, cells from WS patients have a reduced replicative life span in culture . Genomic instability is observed at the cytogenetic level in the form of chromosome breaks and translocations and at the molecular level by multiple large deletions . The Werner syndrome gene (WRN) has recently been cloned . The predicted product is a 1,432-amino-acid protein whose central domain is homologous to members of the RecQ family of DNA helicases . Such homology does not necessarily mean that WRN encodes an active helicase . For example, the Saccharomyces cerevisiae RAD26 gene protein and the human transcription-repair coupling factor CSB (Cockayne syndrome 8) are highly homologous to known helicases, yet neither encodes an active helicase . Moreover, the Bloom's syndrome gene (BLM), discovered before WRN, is also homologous to the RecQ family of DNA helicases, though we still await demonstration that it encodes an active helicase . Here we report that the WS protein does indeed catalyze DNA unwinding.

J Bone Miner Res, 1997 Sep, 12(9), 1348 - 57
Binding and cyclic AMP stimulation by N-terminally deleted human PTHs (3-84 and 4-84) in a homologous ligand receptor system; Olstad OK et al.; We have produced in yeast two human parathyroid hormone (hPTH) analogs with amino-terminal deletions, hPTH(3-84) and hPTH(4-84), employing the mating factor alpha (MF alpha) expression system . The authenticity of the polypeptides was demonstrated by amino-terminal analysis, amino acid composition, and molecular mass analysis . In cells (LLC-PK1) transfected with the human PTH/parathyroid hormone-related protein (PTHrP) receptor, using {125I-Tyr36}chickenPTHrP(1-36)NH2 as radioligand, binding studies revealed dissociation constants at equilibrium (Kd) for hPTH(3-84) and hPTH(4-84) of 4.7 and 8.0 nM, respectively, only slightly higher than natural recombinant hPTH(1-84) Kd = 2.3 nM) . In comparison, {Nle8,18,Tyr34}bovinePTH(3-34)NH2 and {Tyr36}cPTHrP(1-36)NH2 showed equal Kd's of 1.9 nM . Neither of the N-terminally deleted hPTH analogs showed any detectable stimulation of cAMP production in the cells at concentrations below 20 nM . At supersaturated concentrations (500 nM) with receptor occupancy of more than 95% these hPTH analogs revealed about 15% rest agonism compared with that of hPTH(1-84) . hPTH(1-84) and {Tyr36}cPTHrP(1-36)NH2 showed an equal half maximal cyclic adenosine monophosphate (cAMP) stimulation of about 0.8 and 0.7 nM, respectively . The hPTH analogs did not show any ability to antagonize cellular cAMP production induced by either hPTH or {Tyr36}cPTHrP(1-36)NH2 . {Nle8,18,Tyr34}bPTH(3-34)NH2 did also not antagonize cAMP stimulation by hPTH, but inhibited {Tyr36}cPTHrP(1-36)NH2-induced cAMP production by 40% when present at a 1000 M excess . These distinct results related to PTH and PTHrP from different species are important to consider in experiments evaluating potential hPTH or PTHrP antagonism, and employment of a hPTH/PTHrP receptor model is a requirement.

Curr Biol, 1997 Sep 1, 7(9), 689 - 92
Acetylation of general transcription factors by histone acetyltransferases; Imhof A et al.; The acetylation of histones increases the accessibility of nucleosomal DNA to transcription factors {1,2}, relieving transcriptional repression {3} and correlating with the potential for transcriptional activity in vivo {4 - 7} . The characterization of several novel histone acetyltransferases - including the human GCN5 homolog PCAF (p300/CBP-associated factor) {8}, the transcription coactivator p300/CBP {9}, and TAFII250 {10} - has provided a potential explanation for the relationship between histone acetylation and transcriptional activation . In addition to histones, however, other components of the basal transcription machinery might be acetylated by these enzymes and directly affect transcription . Here, we examine the acetylation of the basal transcriptional machinery for RNA polymerase II by PCAF, p300 and TAFII250 . We find that all three acetyltransferases can direct the acetylation of TFIIEbetaand TFIIF, and we identify a preferred site of acetylation in TFIIEbeta . Human TFIIE consists of two subunits, alpha(p56) and beta(p34), which form a heterotetramer (alpha2 beta2) in solution ({11}, reviewed in {12}) . TFIIE enters the preinitiation complex after RNA polymerase II and TFIIF, suggesting that TFIIE may interact directly with RNA polymerase II and/or TFIIF {13,14} . In addition, TFIIE can facilitate promoter melting either in the presence or absence of TFIIH and can stimulate TFIIH-dependent phosphorylation of the carboxy-terminal domain of RNA polymerase II {15-18} . TFIIF has an essential role in both transcription initiation and elongation ({19,20}, for review see {21}) . We discuss the implications of the acetylation of TFIIEbetaand TFIIF for transcriptional control by PCAF, p300 and TAFII250.

Curr Biol, 1997 Sep 1, 7(9), R552 - 5
Protein degradation: the ins and outs of the matter; Cresswell P et al.; In eukaryotic cells, nascent membrane or secretory proteins are translocated into the endoplasmic reticulum through the Sec61p translocation channel; recent evidence suggests that, if they fail to achieve a native conformation, they are translocated back into the cytosol by the same route and degraded by the proteasome.

Curr Biol, 1997 Sep 1, 7(9), R562 - 4
Cell polarity: par for the polar course; Nelson WJ et al.; The nematode PAR-1 gene is required for asymmetric cell divisions during development . Recently identified mammalian Par-1 homologues are kinases that phosphorylate microtubule-associated proteins; their overexpression disrupts the microtubule cytoskeleton, and alters cellular structure and organization.

Mol Biochem Parasitol, 1997 Sep, 88(1-2), 13 - 23
Structure of the Trypanosoma brucei U6 snRNA gene promoter; Nakaar V et al.; Transcription in vivo of small nuclear and cytoplasmic RNA genes of Trypanosoma brucei was previously shown to require the A and B blocks of a divergently transcribed tRNA or tRNA-like gene located approximately 100 nucleotides (nt) upstream . To understand the functioning of these transcription units, we have used the U6 snRNA/tRNA(Thr) genes as a model system . Saturation mutagenesis revealed that for transcription in vivo three elements are essential and sufficient . In addition to the previously described A and B boxes, sequences in the U6 coding region close to the 5' end participate in positioning RNA polymerase III at the start site, and thus constitute a third promoter element . We further showed that the function of the upstream A box, but not the B box, is strictly dependent upon its distance to the U6 gene internal control region . Using our recently developed transcription extract we further demonstrated that in vitro U6 transcription requires only the intragenic sequences and the upstream A box of the tRNA(Thr) gene . This apparent discrepancy between the in vivo and in vitro requirements is highly reminiscent of U6 snRNA gene transcription in the yeast Saccharomyces cerevisiae, and suggests the possibility that similar to the yeast system the B block of the trypanosome U6 snRNA gene promoter might be involved in chromatin organization.

Mol Cell Biol, 1997 Sep, 17(9), 5648 - 55
Dual requirement for a newly identified phosphorylation site in p70s6k; Moser BA et al.; The activation of p70s6k is associated with multiple phosphorylations at two sets of sites . The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1 . The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues . Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites . A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity . Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family . Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism . Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity . Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.

Mol Cell Biol, 1997 Sep, 17(9), 5598 - 611
Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1; Woods D et al.; The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle . Although conditional activation of deltaA-Raf:ER promoted cell cycle progression, activation of deltaRaf-1:ER and deltaB-Raf:ER elicited a G1 arrest that was not overcome by exogenously added growth factors . Activation of all three deltaRaf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27Kip1 . However, activation of deltaB-Raf:ER and deltaRaf-1:ER induced the expression of p21Cip1, whereas activation of deltaA-Raf:ER did not . A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to deltaB-Raf:ER and deltaRaf-1:ER . These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway . By titration of deltaB-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity . Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest . By contrast, activation of Raf in p21Cip1(-/-) MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor . These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts . The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to alpha-factor arrest in Saccharomyces cerevisiae . These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.

Mol Cell Biol, 1997 Sep, 17(9), 5461 - 72
Identification of a member of a DNA-dependent ATPase family that causes interference with silencing; Zhang Z et al.; DNA in eukaryotic cells is packed in tandem repeats of nucleosomes or higher-order chromatin structures, which present obstacles to many cellular processes that require protein-DNA interactions, such as transcription, DNA repair, and recombination . To find proteins that are involved in increasing the accessibility of specific DNA regions in yeast, we used a genetic approach that exploited transcriptional silencing normally occurring at HML and HMR loci . The silencing is mediated by cis-acting silencer elements and is thought to require the formation of a special chromatin structure that prevents accessibility to the silenced DNA . A previously uncharacterized gene, termed DIS1, was isolated from a screen for genes that interfere with silencing when overexpressed . DIS1 encodes a protein with conserved motifs that are present in a family of DNA-dependent ATPases, the SWI2/SNF2-like proteins . Overproduction of N-terminal half of DIS1 protein interfered specifically with ectopic silencing used in the screen as well as HMR E silencing . Two-hybrid studies revealed a specific interaction between the N terminus of DIS1 and the C-terminal half of SIR4, a protein essential for silencing . Cells with a dis1 knockout mutation had significantly lower mating-type switching rate . These results suggest that DIS1 may contribute to making the silenced DNA template at HM loci more accessible during the mating-type switching process.

Mol Cell Biol, 1997 Sep, 17(9), 5299 - 306
Domains of the Brf component of RNA polymerase III transcription factor IIIB (TFIIIB): functions in assembly of TFIIIB-DNA complexes and recruitment of RNA polymerase to the promoter; Kassavetis GA et al.; Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) is composed of three subunits: the TATA-binding protein, the TFIIB-related protein Brf, and B" . TFIIIB, which is brought to RNA polymerase III-transcribed genes indirectly through interaction with DNA-bound TFIIIC or directly through DNA recognition by the TATA-binding protein, in turn recruits RNA polymerase III to the promoter . N-terminally deleted derivatives of Brf have been examined for their ability to interact with DNA-bound TFIIIC and with the other components of TFIIIB and for participation in transcription . Brf(165-596), lacking 164 N-proximal TFIIB-homologous amino acids, is competent to participate in the assembly of TFIIIB-DNA complexes and in TFIIIC-independent transcription . Even deletion of the entire TFIIB-homologous half of the protein, as in Brf(317-596) and Brf(352-596), allows some interaction with DNA-bound TBP and with the B" component of TFIIIB to be retained . The function of Brf(165-596) in transcription has also been examined in the context of B" with small internal deletions . The ability of Brf with this sizable N-terminal segment deleted to function in TFIIIC-independent transcription requires segments of B" that are individually indispensable although required on an either/or basis, in the context of complete Brf . These findings suggest a functional complementarity and reciprocity between the Brf and B" components of TFIIIB.

Mol Cell Biol, 1997 Sep, 17(9), 5136 - 45
Qsr1p, a 60S ribosomal subunit protein, is required for joining of 40S and 60S subunits; Eisinger DP et al.; QSR1 is a recently discovered, essential Saccharomyces cerevisiae gene, which encodes a 60S ribosomal subunit protein . Thirty-one unique temperature-sensitive alleles of QSR1 were generated by regional codon randomization within a conserved 20-amino-acid sequence of the QSR1-encoded protein . The temperature-sensitive mutants arrest as viable, large, unbudded cells 24 to 48 h after a shift to 37 degrees C . Polysome and ribosomal subunit analysis by velocity gradient centrifugation of lysates from temperature-sensitive qsr1 mutants and from cells in which Qsr1p was depleted by down regulation of an inducible promoter revealed the presence of half-mer polysomes and a large pool of free 60S subunits that lack Qsr1p . In vitro subunit-joining assays and analysis of a mutant conditional for the synthesis of Qsr1p demonstrate that 60S subunits devoid of Qsr1p are unable to join with 40S subunits whereas 60S subunits that contain either wild-type or mutant forms of the protein are capable of subunit joining . The defective 60S subunits result from a reduced association of mutant Qsr1p with 60S subunits . These results indicate that Qsr1p is required for ribosomal subunit joining.

Mol Cell Biol, 1997 Sep, 17(9), 5077 - 86
RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein; Ach RA et al.; Unlike mammalian and yeast cells, little is known about how plants regulate G1 progression and entry into the S phase of the cell cycle . In mammalian cells, a key regulator of this process is the retinoblastoma tumor suppressor protein (RB) . In contrast, G1 control in Saccharomyces cerevisiae does not utilize an RB-like protein . We report here the cloning of cDNAs from two Zea mays genes, RRB1 and RRB2, that encode RB-related proteins . Further, RRB2 transcripts are alternatively spliced to yield two proteins with different C termini . At least one RRB gene is expressed in all the tissues examined, with the highest levels seen in the shoot apex . RRB1 is a 96-kDa nuclear protein that can physically interact with two mammalian DNA tumor virus oncoproteins, simian virus 40 large-T antigen and adenovirus E1A, and with a plant D-type cyclin . These associations are abolished by mutation of a conserved cysteine residue in RRB1 that is also essential for RB function . RRB1 binding potential is also sensitive to deletions in the conserved A and B domains, although differences exist in these effects compared to those of human RB . RRB1 can also bind to the AL1 protein from tomato golden mosaic virus (TGMV), a protein which is essential for TGMV DNA replication . These results suggest that G1 regulation in plant cells is controlled by a mechanism which is much more similar to that found in mammalian cells than that in yeast.

Mol Cell Biol, 1997 Sep, 17(9), 5053 - 66
The nuclear orphan receptors COUP-TF and ARP-1 positively regulate the trout estrogen receptor gene through enhancing autoregulation; Lazennec G et al.; The rainbow trout estrogen receptor (rtER) is a positively autoregulated gene in liver cells . In a previous report, we showed that upregulation is mediated by an estrogen response element (ERE) located in the proximal promoter of the gene and that a half binding site for nuclear receptors (5'-TGACCT-3') located 15 bp upstream of the ERE is involved in the magnitude of the estrogen response . We now report that the human orphan receptor COUP-TF and a COUP-TF-like protein from trout liver are able to bind to the consensus half-site . When cotransfected with the rtER gene proximal promoter, COUP-TF had no regulatory functions on its own . Interestingly, COUP-TF enhanced rtER transactivation properties in the presence of estradiol in a dose-dependent manner when cotransfected with the rtER gene promoter . Unliganded retinoid receptor heterodimers had the same helper function as COUP-TF in the presence of estradiol but were switched to repressors when the ligand all-trans-retinoic acid was added . Mutation of the consensus half-site only slightly reduced COUP-TF helper function, suggesting that it actually results from a complex mechanism that probably involves both DNA binding of COUP-TF to the promoter and protein-protein interaction with another transcription factor bound to the promoter . Nevertheless, a DNA-binding-defective mutant of COUP-TF was also defective in ER helper function . Competition footprinting analysis suggested that COUP-TF actually establishes contacts with the consensus upstream half-site and the downstream ERE half-site that would form a DR-24-like response element . Interaction of COUP-TF with the DR-24 element was confirmed in footprinting assays by using nuclear extracts from Saccharomyces cerevisiae expressing COUP-TF . Finally, interaction of COUP-TF with mutants of the rtER gene promoter showed that COUP-TF recognizes the ERE when the upstream half-site is mutated . These data show that COUP-TF may activate transcription through interaction with other nuclear receptors . This cross-talk between liganded nuclear receptors and orphan receptors is likely to modulate the spectrum of action of a particular ligand-receptor complex and may participate in the cell-type specificity of the ligand effect.

Mol Cell Biol, 1997 Sep, 17(9), 4957 - 66
Interaction of ATF6 and serum response factor; Zhu C et al.; Serum response factor (SRF) is a transcription factor which binds to the serum response element (SRE) in the c-fos promoter . It is required for regulated expression of the c-fos gene as well as other immediate-early genes and some tissue-specific genes . To better understand the regulation of SRF, we used a yeast interaction assay to screen a human HeLa cell cDNA library for SRF-interacting proteins . ATF6, a basic-leucine zipper protein, was isolated by binding to SRF and in particular to its transcriptional activation domain . The binding of ATF6 to SRF was also detected in vitro . An ATF6-VP16 chimera activated expression of an SRE reporter gene in HeLa cells, suggesting that ATF6 can interact with endogenous SRF . More strikingly, an antisense ATF6 construct reduced serum induction of a c-fos reporter gene, suggesting that ATF6 is involved in activation of transcription by SRF . ATF6 was previously partially cloned as a member of the ATF family . The complete cDNA of ATF6 was isolated, and its expression pattern was described.

J Virol, 1997 Sep, 71(9), 6842 - 9
Direct repeats of the herpes simplex virus a sequence promote nonconservative homologous recombination that is not dependent on XPF/ERCC4; Yao XD et al.; We have examined mechanisms of recombination in mammalian cells infected with herpes simplex virus type 1 (HSV-1) . Amplification of plasmids containing a viral origin of replication, oriS, in cells superinfected with HSV-1 revealed that linear DNA could be efficiently converted to templates for replication . Two distinct pathways were observed: imprecise end joining and nonconservative homologous recombination . We noted that direct repeats of the viral a sequence promoted efficient nonconservative homologous recombination in BHK cells as well as human repair-proficient 1BR.3N cells and xeroderma pigmentosum group F (XP-F) cells . The reaction gave rise to functional a sequences supporting the formation of defective viruses . It did not seem to proceed by single-strand annealing since it occurred in the absence of XPF/ERCC4, the mammalian homolog of the Rad1 endonuclease from Saccharomyces cerevisiae . In contrast, direct repeats of a 161-bp nonviral sequence did not take part in nonconservative homologous recombination in XP-F cells . Our results suggest that homologous recombination may be involved in the circularization of viral genomes . Furthermore, they demonstrate that amplification of recombination products supported by HSV-1 allows a direct examination of pathways for double-strand-break repair in human cells.

Nucleic Acids Res, 1997 Sep 1, 25(17), 3550 - 1
A solid-phase assay to screen monoclonal antibodies against DNA-binding protein; Ishii N et al.; A method is described for selecting monoclonal antibodies (mAb) against DNA-binding protein . The protocol involves a non-radioactive solid-phase DNA binding assay using a 96-well plate . Because the solid-phase assay is highly specific and sensitive, partially purified antigen is sufficient for the immunization, and mAb screening can be performed with crude cell extract as the antigen . MAbs obtained by this method could supershift the DNA-protein complex in the electromobility shift assay, and were sufficient for immunoscreening of a cDNA expression library.

J Biol Chem, 1997 Aug 29, 272(35), 22340 - 8
Acidic residues necessary for pyrophosphate-energized pumping and inhibition of the vacuolar H+-pyrophosphatase by N,N'-dicyclohexylcarbodiimide; Zhen RG et al.; On the basis of a revised topological model of the vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1) derived from the analysis of four published sequences using two structure-predicting programs, TopPred II and MEMSAT, eight acidic amino acid residues located near or within transmembrane alpha-helices were identified . The codons specifying these amino acids in the cDNA encoding the V-PPase from Arabidopsis thaliana were singly mutated to examine their involvement in pyrophosphate (PPi) hydrolysis and PPi-dependent H+ translocation and the functional significance of the similarities between the sequences encompassing Glu229 (227-245) of the V-PPase and the N,N'-dicyclohexylcarbodiimide (DCCD)-binding transmembrane alpha-helix of the c-subunits of F-ATPases (Nyren, P., Sakai-Nore, Y . , and Strid, A . (1993) Plant Cell Physiol . 34, 375-378) . Three functional classes were identified after heterologous expression of mutated enzyme in Saccharomyces cerevisiae . Class I (E119Q, E229Q, D573N, E667Q, and E751Q) mutants exhibited PPi hydrolytic and H+ translocation activities and DCCD sensitivities similar to wild type . The one class II mutant obtained (E427Q) was preferentially impaired for H+ translocation over PPi hydrolysis but retained sensitivity to DCCD . Class III (E305Q and D504N) mutants exhibited a near complete abolition of both PPi hydrolysis and H+ translocation and residual activities with decreased DCCD sensitivity . In none of the mutants was diminished insertion of the V-PPase into the membrane or an increase in the background conductance of the membrane to H+ evident . The decoupled character of E427Q mutants and the enhancement of H+ pumping in E427D mutants by comparison with wild type, in conjunction with the retention of DCCD inhibitability in both E427Q and E427D mutants, implicate a role for Glu427 in DCCD-insensitive H+ translocation by the V-PPase . The proportionate diminution of PPi hydrolytic and H+ translocation activity and conservation of wild type DCCD sensitivity in E229Q mutants refute the notion that Glu229 is the residue whose covalent modification by DCCD is responsible for the abolition of PPi-dependent H+ translocation . Instead, the diminished sensitivity of the residual activities of E305Q and D504N mutants, but not E305D or D504E mutants, to inhibition by DCCD is consistent with the involvement of acidic residues at these positions in inhibitory DCCD binding . The results are discussed with regard to the possible involvement of Glu427 in coupling PPi hydrolysis with transmembrane H+ translocation and earlier interpretations of the susceptibility of the V-PPase to inhibition by carbodiimides.

Nature, 1997 Aug 28, 388(6645), 891 - 5
Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation; Plemper RK et al.; Proteins enter the secretory pathway through the endoplasmic reticulum, which delivers properly folded proteins to their site of action and contains a quality-control system to monitor and prevent abnormal proteins from being delivered . Many of these proteins are degraded by the cytoplasmic proteasome, which requires their retrograde transport to the cytoplasm . Based on a co-immunoprecipitation of major histocompatibility complex (MHC) class I heavy-chain breakdown intermediates with the translocon subunit Sec61p, it was speculated that Sec61p maybe involved in retrograde transport . Here we present functional evidence from genetic studies that Sec61p mediates retrograde transport of a mutated lumenal yeast carboxypeptidase ycsY (CPY*) in vivo . The endoplasmic reticulum lumenal chaperone BiP (Kar2p) and Sec63p, which are also subunits of the import machinery, are involved in export of CPY* to the cytosol . Thus our results demonstrate that retrograde transport of proteins is mediated by a functional translocon . We consider the export of endoplasmic reticulum-localized proteins to the cytosol by the translocon for proteasome degradation to be a general process in eukaryotic cell biology.

Nature, 1997 Aug 28, 388(6645), 888 - 91
Kinetochores distinguish GTP from GDP forms of the microtubule lattice; Severin FF et al.; During prometaphase in mitotic cell division, chromosomes attach to the walls of microtubules and subsequently move to microtubule ends, where they stay throughout mitosis . This end-attachment seems to be essential for correct chromosome segregating . However, the mechanism by which kinetochores, the multiprotein complexes that link chromosomes to the microtubules of the mitotic spindle, recognize and stay attached to microtubule ends is not understood . One clue comes from the hydrolysis of GTP that occurs during microtubule polymerization . Although tubulin dimers must contain GTP to polymerize, this GTP is rapidly hydrolysed following the addition of dimers to a growing polymer . This creates a microtubule consisting largely of GDP-tubulin, with a small cap of GTP-tubulin at the end . It is possible that kinetochores distinguish the different structural states of a GTP- versus a GDP-microtubule lattice . We have examined this question in vitro using reconstituted kinetochores from the yeast Saccharomyces cerevisiae . We found that kinetochores in vitro bind preferentially to GTP- rather than GDP-microtubules, and to the plus-end preferentially over the lattice . Our results could explain how kinetochores stay at microtubule ends and thus segregate chromosomes correctly during mitosis in vivo . This result demonstrates that proteins exist that can distinguish the GTP conformation of the microtubule lattice.

J Cell Biol, 1997 Aug 25, 138(4), 821 - 32
Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors; Tian G et al.; The production of native alpha/beta tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors . We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on beta-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers . However, this set of cofactors generates native heterodimers only very inefficiently from alpha-tubulin folding intermediates produced by the same chaperonin . Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of alpha-tubulin folding in vitro . This enabled an integrated study of alpha- and beta-tubulin folding: we find that the pathways leading to the formation of native alpha- and beta-tubulin converge in that the folding of the alpha subunit requires the participation of cofactor complexes containing the beta subunit and vice versa . We also show that sequestration of native alpha-or beta-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit . These data demonstrate that tubulin folding cofactors function by placing and/or maintaining alpha-and beta-tubulin polypeptides in an activated conformational state required for the formation of native alpha/beta heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.

J Biol Chem, 1997 Aug 22, 272(34), 21495 - 503
Two-hybrid analysis reveals fundamental differences in direct interactions between desmoplakin and cell type-specific intermediate filaments; Meng JJ et al.; Desmosomes are cell junctions that act as sites of strong intercellular adhesion and also serve to anchor the intermediate filament (IF) cytoskeleton to the plasma membrane of a variety of cell types . Previous studies demonstrated that the COOH terminus of the desmosomal plaque protein, desmoplakin (DP), is required for the association of DP with IF networks in cultured cells and that this domain interacts directly with type II epidermal keratin polypeptides in vitro . However, these studies left open the question of how desmosomes might anchor other IF types known to associate with these junctions . In this report we used yeast two-hybrid and in vitro dot blot assays to further examine the requirements for direct interactions between desmoplakin and various IF types . Our results confirm the ability of the DP COOH terminus (DPCT) to interact with at least two regions of the head domain of the type II epidermal keratin K1 and also demonstrate that DPCT can interact with the type III IF family members, vimentin and desmin, as well as simple epithelial keratins . Unlike the situation for type II epidermal keratins, the interaction between DPCT and simple epithelial keratins appears to depend on heterodimerization of the type I and II keratin polypeptides, since both are required to detect an interaction . Furthermore, although the interaction between DPCT and K1 requires the keratin head domain, deletion of this domain from the simple epithelial keratins does not compromise interaction with DPCT . The interaction between DPCT and type III or simple epithelial keratins also appeared to be less robust than that between DPCT and K1 . In the case of K8/K18, however, the interaction as assessed by yeast two-hybrid assays increased 9-fold when a serine located in a protein kinase A consensus phosphorylation site 23 residues from the end of DP was altered to a glycine . Taken together, these data indicate that DP interacts directly with different IF types in specific ways.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9273 - 8
Interaction of CTLA-4 with AP50, a clathrin-coated pit adaptor protein; Zhang Y et al.; CTLA-4 plays a critical role in regulating the immune response . It is mainly located in cytoplasmic vesicles and is expressed only transiently on the surface after T cell activation . In this study, we demonstrate that CTLA-4 is associated with AP50, the medium chain of the clathrin-associated coated pit adaptor protein complex AP2 . In a yeast two-hybrid screen, three individual cDNA clones that encode mouse AP50 were isolated, all of which can interact specifically with the cytoplasmic domain of mouse CTLA-4, but not with the cytoplasmic domain of mouse CD28 . We have shown that CTLA-4 can bind specifically to AP50 when CTLA-4 and AP50 are cotransfected into human 293T cells . A Y201 to F201 mutation in the YVKM intracellular localization motif of the CTLA-4 cytoplasmic domain significantly diminished its binding to AP50 . We also found that AP50 bound to a CTLA-4 peptide containing unphosphorylated Y201 but not to a peptide containing phosphorylated Y201 . Conversely, the p85 subunit of phosphatidylinositol 3-kinase and, to a lesser extent, protein tyrosine phosphatase SYP (SHP-2) and SHP (SHP-1) bind only to the CTLA-4 peptide containing phosphorylated Y201 . Therefore, the phosphorylation status of Y201 in the CTLA-4 cytoplasmic domain determines the binding specificity of CTLA-4 . These results suggest that AP50 and the coated pit adaptor complex AP2 may play an important role in regulating the intracellular trafficking and function of CTLA-4.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9046 - 51
Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation; Tarun SZ Jr et al.; The yeast translation factor eIF4G associates with both the cap-binding protein eIF4E and the poly(A)-binding protein Pab1p . Here we report that the two yeast eIF4G homologs, Tif4631p and Tif4632p, share a conserved Pab1p-binding site . This site is required for Pab1p and poly(A) tails to stimulate the in vitro translation of uncapped polyadenylylated mRNA, and the region encompassing it is required for the cap and the poly(A) tail to synergistically stimulate translation . This region on Tif4631p becomes essential for cell growth when the eIF4E binding site on Tif4631p is mutated . Pab1p mutations also show synthetic lethal interactions with eIF4E mutations . These data suggest that eIF4G mediates poly(A) tail stimulated translation in vitro, and that Pab1p and the domain encompassing the Pab1p-binding site on eIF4G can compensate for partial loss of eIF4E function in vivo.

Biochemistry, 1997 Aug 19, 36(33), 10082 - 8
Importance of phospholipid in the folding and conformation of phosphatidylinositol transfer protein: comparison of apo and holo species; Voziyan PA et al.; The significance of noncovalently bound phospholipid as a structural component of phosphatidylinositol transfer protein (PITP) and its role in acquisition and maintenance of the native conformation of the protein have been addressed by studying the refolding of PITP after exposure to 6 M guanidinium chloride (GdnCl) . Protein conformations were characterized by (1) the intrinsic tryptophan fluorescence, circular dichroism, and absorbance spectroscopy, (2) the degree of binding of the fluorescent probe 1,8-ANS, and (3) limited proteolytic digestion . When the GdnCl concentration was reduced 100-fold by rapid dilution at 25 degrees C, practically all of the native transfer activity was regained within 20 min . Endogenous phospholipid demonstrated a strong interaction with the native PITP . Separation of the phospholipid from the protein by chromatography on a lipophilic matrix was achieved only under denaturing conditions and resulted in spontaneous oxidation of the apo-protein, accompanied by almost complete loss of recoverable transfer activity . Under reducing conditions, however, apo-PITP recovered more than 80% of the native transfer activity and was similar to holo-PITP in the kinetics of phospholipid transfer . Renatured apo-PITP demonstrated a significant relaxation of the tertiary structure, compared to native and renatured holo-PITP . Incubation of apo-PITP with phospholipid vesicles resulted in a more compact protein conformation . We conclude that the polypeptide can spontaneously fold to a native-like conformation, sufficient for interaction with a lipid membrane and acquisition of a phospholipid ligand . Binding of a phospholipid ligand brings about the final adjustments of protein conformation to the more compact native structure.

Biochemistry, 1997 Aug 19, 36(33), 10026 - 32
DNA bending by GCN4 mutants bearing cationic residues; Strauss-Soukup JK et al.; Transcription activation is thought to require DNA bending to promote the interaction of upstream activators and the basal transcription machinery . Previous experiments have shown that some members of the bZIP family of DNA binding proteins bend DNA, while others do not . We are exploring the possibility that electrostatic effects play a role in these differences . The yeast bZIP transcription factor GCN4 does not induce DNA bending in vitro . Substitution of basic residues for three neutral amino acids of GCN4 confers the ability to bend DNA . This result is consistent with a model of induced DNA bending wherein excess positive charge in proximity to one face of the double helix neutralizes local phosphate diester anions resulting in a laterally asymmetric charge distribution along the DNA . Previous data suggest that such an unbalanced charge distribution results in collapse of the DNA toward the neutralized surface . Interpretations of the present data are discussed . Our result supports the hypothesis that electrostatic interactions can play a key role in DNA bending by bZIP proteins.

Blood, 1997 Aug 15, 90(4), 1482 - 9
Novel interaction of apolipoprotein(a) with beta-2 glycoprotein I mediated by the kringle IV domain; Kochl S et al.; Lipoprotein(a) {Lp(a)}, which has been shown to interact with fibrin(ogen) and other components of the blood clotting cascade, is a major independent risk factor for atherothrombotic disease in humans . The physiological function(s) of Lp(a), as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown . Identification of further potential apo(a)-protein ligands may be crucial to illuminate apo(a)'s function(s) and pathophysiological properties . We used the repetitive apo(a) kringle IV type 2, which is variable in number in apo(a), to screen a human liver cDNA library by the yeast two-hybrid interaction trap system . Among 11 positive clones that emerged from the screen, eight clones were identified as beta-2 glycoprotein I and one as fibronectin . Coimmunoprecipitation experiments confirmed that beta-2 glycoprotein I and apo(a)/Lp(a) interact in human plasma and in cell culture supernatants of COS-1 cells, which ectopically expressed apo(a) . The apo(a)-beta2-glycoprotein I interaction indicates new potential roles for Lp(a) in fibrinolysis and autoimmunity.

Dev Biol, 1997 Aug 15, 188(2), 312 - 21
A role for cyclin E/Cdk2 in the timing of the midblastula transition in Xenopus embryos; Hartley RS et al.; During Xenopus development, the early cell cycles consist of rapid oscillations between DNA synthesis and mitosis until completion of the 12th mitotic division . Then the cycle lengthens and becomes asynchronous, zygotic transcription begins, and G phases are established, a period known as the midblastula transition (MBT) . Some aspects of the MBT, such as zygotic transcription, depend on acquisition of a threshold nuclear to cytoplasmic (N/C) ratio, whereas others, such as maternal cyclin E degradation, are independent of nuclear events and appear to be controlled by an autonomous maternal timer . To investigate the function of cyclin E during the early cycles, cyclin E/Cdk2 kinase activity was specifically inhibited in fertilized eggs by a truncated form of the Xenopus Cdk inhibitor, Xic1 (Delta34Xic1) . Delta34Xic1 caused lengthening of the embryonic cell cycles that correlated with increased levels of mitotic cyclins . However, DNA synthesis was not inhibited . Several hallmarks of the MBT were delayed for several hours in Delta34Xic1-injected embryos, including the disappearance of cyclins E and A, the initiation of zygotic transcription, and the reappearance of phosphotyrosine on Cdc2 . In both control and Delta34Xic1-injected embryos, cyclin E was degraded after the 12th mitotic division as zygotic transcription began, but experiments with alpha-amanitin show that cyclin E degradation is not dependent on zygotic transcription . Thus, the length of the early cycles and the timing of maternal cyclin degradation depend upon cyclin E/Cdk2 activity . Neither oscillations in cyclin E/Cdk2 activity during the early cycles nor the disappearance of cyclin E at the MBT were dependent on protein synthesis . These data suggest that cyclin E/Cdk2 is directly linked to an autonomous maternal timer that drives the early embryonic cell cycles until the MBT.

Nucleic Acids Res, 1997 Aug 15, 25(16), 3373 - 4
Improved efficiency sos recruitment system: expression of the mammalian GAP reduces isolation of Ras GTPase false positives; Aronheim A; The Sos recruitment system (SRS) is a novel genetic method for detecting protein-protein interactions . The method is based on localizing Sos, a Ras guanyl nucleotide exchange factor (GEF), to the plasma membrane through interaction between two fusion proteins . Mammalian Ras can bypass the requirement for a functional Ras GEF and represents a predictable false positive in this system . This report demonstrates that introduction of mammalian GTPase activating protein (mGAP) reduces the isolation of Ras false positives in SRS screens of mammalian cDNA libraries, thereby significantly enhancing the efficiency of the system.

Nucleic Acids Res, 1997 Aug 15, 25(16), 3297 - 301
A new method to monitor the rate of conformational transitions in RNA; Maglott EJ et al.; Many RNAs need Mg2+to produce stable tertiary structures . Here we describe a simple method to measure the rate and activation parameters of tertiary structure unfolding that exploits this Mg2+dependence . Our approach is based on mixing an RNA solution with excess EDTA in a stopped-flow instrument equipped with an absorbance detector, under conditions of temperature and ionic strength where, after chelation of Mg2+, tertiary structure unfolds . We have demonstrated the utility of this method by studying phenylalanine-specific transfer RNA from yeast (tRNAPhe) because the unfolding rates and the corresponding activation parameters have been determined previously and provide a benchmark for our technique . We find that within error, our stopped-flow method reproduces both the rate and activation enthalpy for tertiary unfolding of yeast tRNAPhe measured previously by temperature-jump relaxation kinetics . Since many different RNAs require divalent magnesium for tertiary structure stabilization, this technique should be applicable to study the folding of other RNAs.

Oncogene, 1997 Aug 14, 15(7), 807 - 16
Two tandem and independent sub-activation domains in the amino terminus of p53 require the adaptor complex for activity; Candau R et al.; The ability of p53 to function as a tumor suppressor is linked to its function as a transcriptional activator, since p53 mutants that do not transactivate are unable to suppress tumor cell growth . Previous studies identified an activation domain in the amino terminal 40 residues of the protein, a region that binds to several general transcription factors and to some oncogene products . For example, mdm-2, a cellular oncoprotein, binds to this region and represses p53 transactivation . Here we describe a new activation domain within the amino terminus of p53 that maps between amino acids 40-83, and whose residues trp-53 and phe-54 are critical for function both in yeast and in mammalian cells . In vivo studies in yeast show that the new activation subdomain, unlike the previously described, is mdm-2 independent . Both p53 activation subdomains (1-40 and 40-83) require the yeast adaptor complex ADA2/ADA3/GCN5 for transcriptional activation . Moreover, since activation by p53 requires GCN5's enzymatic histone acetyltransferase domain, p53 may regulate gene expression by influencing chromatin modification.

FEBS Lett, 1997 Aug 11, 413(1), 135 - 41
Mutational analysis of human prothymosin alpha reveals a bipartite nuclear localization signal; Rubtsov YP et al.; Mutants of human prothymosin alpha with impaired ability to inhibit yeast Saccharomyces cerevisiae . cerevisiae cell growth were characterized . Two types of prothymosin alpha-inactivating mutations were observed . Mutations that belong to the first type compromised the nuclear entry of prothymosin alpha by affecting its nuclear localization signal . Analysis of subcellular distribution of GFP-prothymosin alpha fusions revealed a bipartite nuclear localization signal that is both necessary and sufficient for nuclear import of the protein in human cells . Mutations of the second type abrogated the inhibitory action of prothymosin alpha through an unknown mechanism, without influencing the nuclear import of the protein.

J Cell Biol, 1997 Aug 11, 138(3), 531 - 45
The membrane protein alkaline phosphatase is delivered to the vacuole by a route that is distinct from the VPS-dependent pathway; Piper RC et al.; Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants . Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles . Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment . The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27 . In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes . Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole . Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole . In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

J Cell Biol, 1997 Aug 11, 138(3), 517 - 29
A multispecificity syntaxin homologue, Vam3p, essential for autophagic and biosynthetic protein transport to the vacuole; Darsow T et al.; Protein transport in eukaryotic cells requires the selective docking and fusion of transport intermediates with the appropriate target membrane . t-SNARE molecules that are associated with distinct intracellular compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions . VAM3 encodes a 283-amino acid protein that shares homology with the syntaxin family of t-SNARE molecules . Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation . Null mutants of vam3 exhibited defects in the maturation of multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments . To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3(tsf)) . Upon shifting the vam3(tsf) mutant cells to nonpermissive temperature, an immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed: transport via both the carboxypeptidase Y pathway and the alkaline phosphatase pathway was inhibited . In addition, vam3(tsf) cells also exhibited defects in autophagy . Both the delivery of aminopeptidase I and the docking/ fusion of autophagosomes with the vacuole were defective at high temperature . Upon temperature shift, vam3(tsf) cells accumulated novel membrane compartments, including multivesicular bodies, which may represent blocked transport intermediates . Genetic interactions between VAM3 and a SEC1 family member, VPS33, suggest the two proteins may act together to direct the docking and/or fusion of multiple transport intermediates with the vacuole . Thus, Vam3p appears to function as a multispecificity receptor in heterotypic membrane docking and fusion reactions with the vacuole . Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3Delta mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12Delta mutant phenotypes . This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.

J Cell Biol, 1997 Aug 11, 138(3), 485 - 94
Mutational analysis of Mdm1p function in nuclear and mitochondrial inheritance; Fisk HA et al.; Nuclear and mitochondrial transmission to daughter buds of Saccharomyces cerevisiae depends on Mdm1p, an intermediate filament-like protein localized to numerous punctate structures distributed throughout the yeast cell cytoplasm . These structures disappear and organelle inheritance is disrupted when mdm1 mutant cells are incubated at the restrictive temperature . To characterize further the function of Mdm1p, new mutant mdm1 alleles that confer temperature-sensitive growth and defects in organelle inheritance but produce stable Mdm1p structures were isolated . Microscopic analysis of the new mdm1 mutants revealed three phenotypic classes: Class I mutants showed defects in both mitochondrial and nuclear transmission; Class II alleles displayed defective mitochondrial inheritance but had no effect on nuclear movement; and Class III mutants showed aberrant nuclear inheritance but normal mitochondrial distribution . Class I and II mutants also exhibited altered mitochondrial morphology, possessing primarily small, round mitochondria instead of the extended tubular structures found in wild-type cells . Mutant mdm1 alleles affecting nuclear transmission were of two types: Class Ia and IIIa mutants were deficient for nuclear movement into daughter buds, while Class Ib and IIIb mutants displayed a complete transfer of all nuclear DNA into buds . The mutations defining all three allelic classes mapped to two distinct domains within the Mdm1p protein . Genetic crosses of yeast strains containing different mdm1 alleles revealed complex genetic interactions including intragenic suppression, synthetic phenotypes, and intragenic complementation . These results support a model of Mdm1p function in which a network comprised of multimeric assemblies of the protein mediates two distinct cellular processes.

J Biol Chem, 1997 Aug 8, 272(32), 19641 - 4
CASH, a novel caspase homologue with death effector domains; Goltsev YV et al.; CASP-8 and CASP-10, members of a cysteine protease family that participates in apoptosis, interact with MORT1/FADD, an adapter protein in the CD120a (p55 tumor necrosis factor receptor), and CD95 (Fas/Apo-1) death-inducing signaling pathways, through a shared N-terminal sequence motif, the death effector domain . We report cloning of two splice variants of a novel protein, CASH, that contain two N-terminal death effector domains and can bind through them to each other, to MORT1/FADD, to CASP-8, and to CASP-10 . The unique C-terminal part of the longer variant shows marked sequence homology to the caspase protease region yet lacks several of the conserved caspase active site residues, suggesting that it is devoid of cysteine protease activity . Overexpression of the short CASH splice variant strongly inhibited cytotoxicity induction by CD120a and CD95 . Expression of the longer variant, while inhibiting cytotoxicity in HeLa cells, had a marked cytocidal effect in 293 cells that could be shown to involve its protease homology region . The findings suggest that CASH acts as an attenuator and/or initiator in CD95 and CD120a signaling for cell death.

J Biol Chem, 1997 Aug 8, 272(32), 19637 - 40
Novel Ca2+-binding protein (CAPS) related to UNC-31 required for Ca2+-activated exocytosis; Ann K et al.; Exocytotic secretion in neuroendocrine cells is activated by cytoplasmic Ca2+ increases . Late post-docking events in dense core vesicle exocytosis in permeable PC12 cells require cytosolic factors for sequential ATP-dependent priming and Ca2+-dependent triggering steps . The cytosolic proteins phosphatidylinositol transfer protein and phosphatidylinositol (4)-phosphate 5-kinase, as well as membrane-bound N-ethylmaleimide-sensitive factor, are required for the ATP-dependent priming step . Following priming, the Ca2+-dependent triggering of vesicle fusion requires an additional cytosolic factor, CAPS, which was purified as a 145-kDa protein . To clarify late Ca2+-dependent events in vesicle fusion, the sequence of rat CAPS cDNA was determined and found to encode a novel protein that is the vertebrate homologue of the Caenorhabditis elegans UNC-31 protein shown genetically to be required for neurosecretion . Recombinant CAPS substituted for cytosol in the Ca2+ triggering step in permeable PC12 cells and exhibited moderate affinity (Kd = 270 microM) Ca2+ binding (2 mol Ca2+/mol CAPS dimer), consistent with a role at a Ca2+-regulated step in exocytosis.

Cell, 1997 Aug 8, 90(3), 569 - 80
Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300; Chen H et al.; We report here the identification of a novel cofactor, ACTR, that directly binds nuclear receptors and stimulates their transcriptional activities in a hormone-dependent fashion . ACTR also recruits two other nuclear factors, CBP and P/CAF, and thus plays a central role in creating a multisubunit coactivator complex . In addition, and unexpectedly, we show that purified ACTR is a potent histone acetyltransferase and appears to define a distinct evolutionary branch to this recently described family . Thus, hormonal activation by nuclear receptors involves the mutual recruitment of at least three classes of histone acetyltransferases that may act cooperatively as an enzymatic unit to reverse the effects of histone deacetylase shown to be part of the nuclear receptor corepressor complex.

Cell, 1997 Aug 8, 90(3), 449 - 58
Miranda is required for the asymmetric localization of Prospero during mitosis in Drosophila; Shen CP et al.; Asymmetric division of Drosophila neuroblasts, sensory organ precursor cells, and cells in the procephalic neurogenic region involves the segregation of Numb and Prospero proteins into one of the two daughter cells . We have isolated a novel gene, miranda, based on the ability of its gene product to interact with the Prospero asymmetric localization domain . miranda expression coincides spatially and temporally with asymmetric cell divisions and asymmetric localization of Prospero . Miranda protein is localized asymmetrically, along with Prospero, to the basal cell membrane during mitosis . Loss of miranda gene function abolishes asymmetric Prospero localization during mitosis . The asymmetric localization of Miranda protein requires inscuteable . Our results suggest that miranda functions downstream of inscuteable and works as an adapter that connects Prospero to the basal cell membrane during asymmetric cell division.

Biochem Biophys Res Commun, 1997 Aug 8, 237(1), 152 - 7
Primary structure of human PMP69, a putative peroxisomal ABC-transporter; Holzinger A et al.; We have cloned the cDNA of a novel human ABC-half-transporter highly similar to peroxisomal ABC-half-transporters such as the adrenoleukodystrophy protein (ALDP) and the peroxisomal protein 70 (PMP70) . This 2927-bp cDNA codes for a 606 aminoacid (68.6 kDa) protein that was designated PMP69 (putative peroxisomal membrane protein) . PMP69 is ubiquitously expressed . Transcript variants resulting from alternative polyadenylation and splicing events including one that confers an alternative C-terminus have been found . The PMP69 gene is localized on chromosome 14q24.3 . ABC-half-transporters require a partner ABC-half-transporter to constitute a functional complex, either as a homodimer or a heterodimer . Defects in the gene coding for ALDP are the cause of adrenoleukodystrophy, a demyelinating disorder of the nervous system with strikingly varying clinical courses . PMP70 was implicated in the pathogenesis of a subgroup of Zellweger syndrome, a heterogenous group of peroxisome assembly disorders . PMP69 might be a heterodimer partner for one of these proteins, thus playing a role in modifying the clinical course of ALD or, alternatively, in peroxisome biogenesis.

J Biol Chem, 1997 Aug 8, 272(32), 20230 - 5
Ligand-independent activation domain in the N terminus of peroxisome proliferator-activated receptor gamma (PPARgamma) . Differential activity of PPARgamma1 and -2 isoforms and influence of insulin; Werman A et al.; Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear hormone receptor superfamily, and is an important regulator of adipogenesis and adipocyte gene expression . PPARgamma exists as two isoforms, PPARgamma1 and PPARgamma2, that differ only in their N termini . Both isoforms are activated by ligands that include the antidiabetic thiazoladinedione drugs and 15-deoxy-Delta12, 14-prostaglandin J2, and potential differences in their function have yet to be described . We report that, in addition to a ligand-activated transcriptional activity, when studied under conditions of ligand depletion, intact PPARgamma has a ligand-independent activation domain . To identify the basis for this ligand-independent activation, we used GAL4-PPARgamma chimeric expression constructs and UAS-TK-LUC in CV1 cells and isolated rat adipocytes . In both cell systems, isolated PPARgamma1 and PPARgamma2 N termini have activation domains, and the activation function of PPARgamma2 is 5-6-fold greater than that of PPARgamma1 . Insulin enhances the transcriptional effect mediated by both PPARgamma1 and PPARgamma2 N-terminal domains . These data demonstrate that 1) PPARgamma has an N-terminal (ligand-independent) activation domain; 2) PPARgamma1 and PPARgamma2 N termini have distinct activation capacities; and 3) insulin can potentiate the activity of the N-terminal domain of PPARgamma.

J Biol Chem, 1997 Aug 8, 272(32), 20146 - 51
Biosynthesis of archaeosine, a novel derivative of 7-deazaguanosine specific to archaeal tRNA, proceeds via a pathway involving base replacement on the tRNA polynucleotide chain; Watanabe M et al.; Archaeosine is a novel derivative of 7-deazaguanosine found in transfer RNAs of most organisms exclusively in the archaeal phylogenetic lineage and is present in the D-loop at position 15 . We show that this modification is formed by a posttranscriptional base replacement reaction, catalyzed by a new tRNA-guanine transglycosylase (TGT), which has been isolated from Haloferax volcanii and purified nearly to homogeneity . The molecular weight of the enzyme was estimated to be 78 kDa by SDS-gel electrophoresis . The enzyme can insert free 7-cyano-7-deazaguanine (preQ0 base) in vitro at position 15 of an H . volcanii tRNA T7 transcript, replacing the guanine originally located at that position without breakage of the phosphodiester backbone . Since archaeosine base and 7-aminomethyl-7-deazaguanine (preQ1 base) were not incorporated into tRNA by this enzyme, preQ0 base appears to be the actual substrate for the TGT of H . volcanii, a conclusion supported by characterization of preQ0 base in an acid-soluble extract of H . volcanii cells . Thus, this novel TGT in H . volcanii is a key enzyme for the biosynthetic pathway leading to archaeosine in archaeal tRNAs.

J Biol Chem, 1997 Aug 8, 272(32), 19801 - 7
Rtg3p, a basic helix-loop-helix/leucine zipper protein that functions in mitochondrial-induced changes in gene expression, contains independent activation domains; Rothermel BA et al.; Rtg3p and Rtg1p are basic helix-loop-helix/leucine zipper protein transcription factors in yeast that interact and bind to sites in an upstream activation sequence element in the 5'-flanking region of CIT2, a gene encoding a peroxisomal isoform of citrate synthase . These factors are required both for basal expression of CIT2 and its elevated expression in cells with dysfunctional mitochondria, such as in respiratory-deficient petite cells lacking mitochondrial DNA (rho degrees ) . This elevated expression of CIT2 is called the retrograde response . Here we show that fusion constructs between the Gal4p DNA binding domain and Rtg3p transactivate the expression of a LacZ reporter gene under the control of a GAL1 promoter element . We have identified two activation domains in Rtg3p: a strong carboxyl-terminal domain from amino acids 375-486, and a weaker amino-terminal domain from amino acids 1-175; neither of these activation domains contain the bHLH/Zip motif . We have also identified a serine/threonine-rich domain of Rtg3p within amino acids 176-282 that is inhibitory to transactivation . In addition, the transcriptional activity of the Gal4-Rtg3p fusion proteins does not require either Rtg1p or Rtg2p; the latter is a protein containing an hsp70-like ATP binding domain that is also necessary for CIT2 expression . In contrast, transcriptional activation by Gal4-Rtg1p fusion proteins requires the Rtg1p basic helix-loop-helix/leucine zipper protein domain, as well as Rtg3p and Rtg2p . These data suggest that transcriptional activation by the Rtg1p-Rtg3p complex is largely the function of Rtg3p . Experiments are also presented suggesting that Rtg3p is limiting for gene expression in respiratory-competent (rho+) cells.

J Biol Chem, 1997 Aug 8, 272(32), 19785 - 93
Differential interactions of Id proteins with basic-helix-loop-helix transcription factors; Langlands K et al.; Dimerization of three Id proteins (Id1, Id2, and Id3) with the four class A E proteins (E12, E47, E2-2, and HEB) and two groups of class B proteins, the myogenic regulatory factors (MRFs: MyoD, myogenin, Myf-5 and MRF4/Myf-6), and the hematopoietic factors (Scl/Tal-1, Tal-2, and Lyl-1) were tested in a quantitative yeast 2-hybrid assay . All three Ids bound with high affinity to E proteins, but a much broader range of interactions was observed between Ids and the class B factors . Id1 and Id2 interacted strongly with MyoD and Myf-5 and weakly with myogenin and MRF4/Myf-6, whereas Id3 interacted weakly with all four MRFs . Similar specificities were observed in co-immunoprecipitation and mammalian 2-hybrid analyses . No interactions were found between the Ids and any of the hematopoietic factors . Each Id was able to disrupt the ability of E protein-MyoD complexes to transactivate from a muscle creatine kinase reporter construct in vivo . Finally, mutagenesis experiments showed that the differences between Id1 and Id3 binding map to three amino acids in the first helix and to a small cluster of upstream residues . The Id proteins thus display a signature range of interactions with all of their potential dimerization partners and may play a role in myogenesis which is distinct from that in hematopoiesis.

Nature, 1997 Aug 7, 388(6642), 593 - 8
Rim is a putative Rab3 effector in regulating synaptic-vesicle fusion; Wang Y et al.; Rab3 is a neuronal GTP-binding protein that regulates fusion of synaptic vesicles and is essential for long-term potentiation of hippocampal mossy fibre synapses . More than thirty Rab GTP-binding proteins are known to function in diverse membrane transport pathways, although their mechanisms of action are unclear . We have now identified a putative Rab3-effector protein called Rim . Rim is composed of an amino-terminal zinc-finger motif and carboxy-terminal PDZ and C2 domains . It binds only to GTP (but not to GDP)-complexed Rab3, and interacts with no other Rab protein tested . There is enrichment of Rab3 and Rim in neurons, where they have complementary distributions . Rab3 is found only on synaptic vesicles, whereas Rim is localized to presynaptic active zones in conventional synapses, and to presynaptic ribbons in ribbon synapses . Transfection of PC12 cells with the amino-terminal domains of Rim greatly enhances regulated exocytosis in a Rab3-dependent manner . We propose that Rim serves as a Rab3-dependent regulator of synaptic-vesicle fusion by forming a GTP-dependent complex between synaptic plasma membranes and docked synaptic vesicles.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8590 - 5
Importin/karyopherin protein family members required for mRNA export from the nucleus; Seedorf M et al.; The yeast Saccharomyces cerevisiae contains three proteins (Kap104p, Pse1p, and Kap123p) that share similarity to the 95-kDa beta subunit of the nuclear transport factor importin (also termed karyopherin and encoded by KAP95/RSL1 in yeast) . Proteins that contain nuclear localization sequences are recognized in the cytoplasm and delivered to the nucleus by the heterodimeric importin complex . A second importin-related protein, transportin, delivers a subset of heterogeneous nuclear ribonucleoproteins (hnRNPs) to the nucleoplasm . We now show that in contrast to loss of importin beta (Kap95p/Rsl1p) and transportin (Kap104p), conditional loss of Pse1p in a strain lacking Kap123p results in a specific block of mRNA export from the nucleus . Overexpression of Sxm1p, a protein related to Cse1p in yeast and to the human cellular apoptosis susceptibility protein, relieves the defects of cells lacking Pse1p and Kap123p . Thus, a major role of Pse1p, Kap123p, and Sxm1p may be nuclear export rather than import, suggesting a symmetrical relationship between these processes.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8569 - 74
The SH3p4/Sh3p8/SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain; Ringstad N et al.; The GTPase dynamin I and the inositol 5-phosphatase synaptojanin are nerve terminal proteins implicated in synaptic vesicle recycling . Both proteins contain COOH-terminal proline-rich domains that can interact with a variety of Src homology 3 (SH3) domains . A major physiological binding partner for dynamin I and synaptojanin in the nervous system is amphiphysin I, an SH3 domain-containing protein also concentrated in nerve terminals . We have used the proline-rich tail of synaptojanin to screen a rat brain library by the two-hybrid method to identify additional interacting partners of synaptojanin . Three related proteins containing SH3 domains that are closely related to the SH3 domains of Grb2 were isolated: SH3p4, SH3p8, and SH3p13 . Further biochemical studies demonstrated that the SH3p4/8/13 proteins bind to both synaptojanin and dynamin I . The SH3p4/8/13 transcripts are differentially expressed in tissues: SH3p4 mRNA was detected only in brain, SH3p13 mRNA was present in brain and testis, and the SH3p8 transcript was detected at similar levels in multiple tissues . Members of the SH3p4/8/13 protein family were found to be concentrated in nerve terminals, and pools of synaptojanin and dynamin I were coprecipitated from brain extracts with antibodies recognizing SH3p4/8/13 . These findings underscore the important role of SH3-mediated interactions in synaptic vesicle recycling.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8521 - 6
In vitro selection of a 7-methyl-guanosine binding RNA that inhibits translation of capped mRNA molecules; Haller AA et al.; Using systematic evolution of ligands by exponential enrichment (SELEX), an RNA molecule was isolated that displays a 1,000-fold higher affinity for guanosine residues that carry an N-7 methyl group than for nonmethylated guanosine residues . The methylated guanosine residue closely resembles the 5' terminal cap structure present on all eukaryotic mRNA molecules . The cap-binding RNA specifically inhibited the translation of capped but not uncapped mRNA molecules in cell-free lysates prepared from either human HeLa cells or from Saccharomyces cerevisiae . These findings indicate that the cap-binding RNA will also be useful in studies of other cap-dependent processes such as pre-mRNA splicing and nucleocytoplasmic mRNA transport.

FEBS Lett, 1997 Aug 4, 412(3), 583 - 6
Activation of transcription factor NF-kappa B by phagocytic stimuli in human neutrophils; McDonald PP et al.; Phagocytosis represents an important physiological trigger for the inducible expression of several genes in human neutrophils . Here, we report that a DNA-binding activity primarily consisting of the classical NF-kappa B heterodimer, p50/RelA, is induced in phagocytosing neutrophils . Under these conditions, NF-kappa B activation was found to be a rapid and transient response, reaching a maximum by 10-15 min, and returning to near-basal levels by 30 min . In neutrophils undergoing the phagocytosis of opsonized yeasts, the onset of NF-kappa B activation was paralleled by a decline in immunoreactive I kappa B-alpha protein levels, and the cellular I kappa B-alpha pool was replenished by 30 min, in agreement with our gel shift data . We conclude that NF-kappa B activation could constitute one of the mechanisms whereby the expression of kappa B-responsive genes is enhanced in phagocytosing neutrophils . To our knowledge, this represents the first demonstration that phagocytic stimuli can induce NF-kappa B activation in human neutrophils.

FEBS Lett, 1997 Aug 4, 412(3), 446 - 52
The Arabidopsis Cks1At protein binds the cyclin-dependent kinases Cdc2aAt and Cdc2bAt; De Veylder L et al.; In Arabidopsis, two cyclin-dependent kinases (CDK), Cdc2aAt and Cdc2bAt, have been described . Here, we have used the yeast two-hybrid system to identify Arabidopsis proteins interacting with Cdc2aAt . Three different clones were isolated, one of which encodes a Suc1/Cks1 homologue . The functionality of the Arabidopsis Suc1/Cks1 homologue, designed Cks1At, was demonstrated by its ability to rescue the temperature-sensitive cdc2-L7 strain of fission yeast at low and intermediate expression levels . In contrast, high cks1At expression levels inhibited cell division in both mutant and wild-type yeast strains . Cks1At binds both Cdc2aAt and Cdc2bAt in vivo and in vitro . Furthermore, we demonstrate that the fission yeast Suc1 binds Cdc2aAt but only weakly Cdc2bAt, whereas the human CksHs1 associated exclusively with Cdc2aAt.

Eur J Biochem, 1997 Aug 1, 247(3), 914 - 9
Fourier transform infrared spectroscopic studies of proton transfer processes and the dissociation of Zn2+-bound water in alcohol dehydrogenases; Nadolny C et al.; The following complexes were investigated by Fourier transform difference spectroscopy: binary complexes of alcohol dehydrogenases from yeast (YADH) and horse liver (LADH) with nicotinamide adenine dinucleotide (NAD+) and adenosine (5')-diphospho(5)-beta-D-ribose (ADP-Rib); the binary complex of Zn2+-free YADH with NAD+, the ternary complex of LADH with NAD+ and 2,2,2-trifluoroethanol . After addition of NAD+ to YADH and LADH, protonation of the N1 atom of the adenine ring of NAD+ is observed . It is shown that this proton arises from the dissociation of the Zn2+-bound water . The interaction of the Zn2+ ion with water is very strong, since this interaction is not just an electrostatic interaction . If the Zn2+ ions are in a tetrahedral environment, a large covalent contribution also occurs . If ADP-Rib is present instead of NAD+, no protonation of the N1 atom of the adenine ring of ADP-Rib is found, which demonstrates that the positively charged nicotinamide ring favors the conduction of the positive charge . All these results confirm the mechanism of Branden et al . (1975): the Zn2+-bound water is split and the arising (OH)- deprotonates the alcohol . In the case of the ternary complex of LADH with NAD+ and 2,2,2-trifluoroethanol, we demonstrate that the alcohol is deprotonated and the alcoholate ion is bound directly to the Zn2+ ion . The conduction of the proton from the active site to the N1 atom of adenine occurs via a hydrogen-bonded chain with large proton polarizability due to collective proton motion . The nature and mechanism of this pathway are discussed on the basis of data from previous studies.

Eur J Biochem, 1997 Aug 1, 247(3), 860 - 9
Determination of functional domains of the human transcription factor PAX8 responsible for its nuclear localization and transactivating potential; Poleev A et al.; The conserved structure of the transcription factors of the Pax gene family may reflect functional conservation . We have demonstrated that the human Pax8 transcription factor is organized in several functional domains and contains two regions responsible for its nuclear localization, in addition to an activating region at the carboxy terminus of the protein and an inhibitory region encoded by the exon 9 present only in a splice variant PAX8a . Regions of PAX8 determining the nuclear localization of the PAX8A/lacZ fusions contain short amino acid sequences similar to several described nuclear localization sites (NLS) . These NLS were identified in the paired domain and between the octapeptide and the residual homeodomain, respectively . The activating domain is encoded by the exons 10 and 11 and its function is modulated by the adjacent domains encoded by the exons 9 and 12 . The domain encoded by exon 9 significantly inhibits the function of the activating domain . Pax8 is expressed in thyroid cells and its product binds promoters of the thyroglobulin and thyroperoxidase genes through its paired domain . Thyroid cell growth and differentiation depend on thyrotropin which, by stimulating cAMP synthesis, activates the cAMP-dependent protein kinase A (PKA) . We have investigated a link between thyrotropin stimulation and gene activation by Pax8 . Stimulation of cAMP synthesis augments Pax8-specific transcription in thyroid cells, indicating that PKA is involved in Pax8 activation . Cotransfection of GAL4/PAX8 fusions and the catalytic subunit of PKA in A126, a PKA-deficient derivative of the PC12 pheochromocytoma cell line, synergistically activates the GAL4-specific reporter, suggesting the activating domain of PAX8 is dependent upon the catalytic subunit of the PKA . We propose that this dependence is due to a hypothetical adaptor which forms a target for PKA and interacts with the activating domain of PAX8 . We show that PAX8 isolated from the thyroid cell line FTRL5 is a phosphoprotein in which phosphorylation is not dependant on cAMP pathway activation . Our results suggest that Pax8 is part of the cAMP signaling pathway and mediates thyrotropin-dependent gene activation in thyroid cells . Investigation of the PAX8 expression in a panel of Wilms' tumors shows a striking correlation between the expression of PAX8 and another transcription factor, WT1, indicating that these two genes may interact in vivo.

Mol Biol Cell, 1997 Aug, 8(8), 1603 - 18
Regulated nuclear translocation of the Mig1 glucose repressor; De Vit MJ et al.; Glucose represses the transcription of many genes in bakers yeast (Saccharomyces cerevisiae) . Mig1 is a Cys2-His2 zinc finger protein that mediates glucose repression of several genes by binding to their promoters and recruiting the general repression complex Ssn6-Tup1 . We have found that the subcellular localization of Mig1 is regulated by glucose . Mig1 is imported into the nucleus within minutes after the addition of glucose and is just as rapidly transported back to the cytoplasm when glucose is removed . This regulated nuclear localization requires components of the glucose repression signal transduction pathway . An internal region of the protein separate from the DNA binding and repression domains is necessary and sufficient for glucose-regulated nuclear import and export . Changes in the phosphorylation status of Mig1 are coincident with the changes in its localization, suggesting a possible regulatory role for phosphorylation . Our results suggest that a glucose-regulated nuclear import and/or export mechanism controls the activity of Mig1.

Mol Biol Cell, 1997 Aug, 8(8), 1529 - 41
A sorting nexin-1 homologue, Vps5p, forms a complex with Vps17p and is required for recycling the vacuolar protein-sorting receptor; Horazdovsky BF et al.; A number of the Saccharomyces cerevisiae vacuolar protein-sorting (vps) mutants exhibit an altered vacuolar morphology . Unlike wild-type cells that contain 1-3 large vacuolar structures, the class B vps5 and vps17 mutant cells contain 10-20 smaller vacuole-like compartments . To explore the role of these VPS gene products in vacuole biogenesis, we cloned and sequenced VPS5 and characterized its protein products . The VPS5 gene is predicted to encode a very hydrophilic protein of 675 amino acids that shows significant sequence homology with mammalian sorting nexin-1 . Polyclonal antiserum directed against the VPS5 gene product detects a single, cytoplasmic protein that is phosphorylated specifically on a serine residue(s) . Subcellular fractionation studies indicate that Vps5p is associated peripherally with a dense membrane fraction distinct from Golgi, endosomal, and vacuolar membranes . This association was found to be dependent on the presence of another class B VPS gene product, Vps17p . Biochemical cross-linking studies demonstrated that Vps5p and Vps17p physically interact . Gene disruption experiments show that the VPS5 genes product is not essential for cell viability; however, cells carrying the null allele contain fragmented vacuoles and exhibit defects in vacuolar protein-sorting similar to vps17 null mutants . More than 95% of carboxypeptidase Y is secreted from these cells in its Golgi-modified p2 precursor form . Additionally, the Vps10p vacuolar protein-sorting receptor is mislocalized to the vacuole in vps5 mutant cells . On the basis of these and other observations, we propose that the Vps17p protein complex may participate in the intracellular trafficking of the Vps10p-sorting receptor, as well as other later-Golgi proteins.

Mol Biol Cell, 1997 Aug, 8(8), 1449 - 60
Membrane translocation of mitochondrially coded Cox2p: distinct requirements for export of N and C termini and dependence on the conserved protein Oxa1p; He S et al.; To study in vivo the export of mitochondrially synthesized protein from the matrix to the intermembrane space, we have fused a synthetic mitochondrial gene, ARG8m, to the Saccharomyces cerevisiae COX2 gene in mitochondrial DNA . The Arg8mp moiety was translocated through the inner membrane when fused to the Cox2p C terminus by a mechanism dependent on topogenic information at least partially contained within the exported Cox2p C-terminal tail . The pre-Cox2p leader peptide did not signal translocation . Export of the Cox2p C-terminal tail, but not the N-terminal tail, was dependent on the inner membrane potential . The mitochondrial export system does not closely resemble the bacterial Sec translocase . However, normal translocation of both exported domains of Cox2p was defective in cells lacking the widely conserved inner membrane protein Oxa1p.

Mol Biol Cell, 1997 Aug, 8(8), 1427 - 37
SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities; Verma R et al.; Traversal from G1 to S-phase in cycling cells of budding yeast is dependent on the destruction of the S-phase cyclin/CDK inhibitor SIC1 . Genetic data suggest that SIC1 proteolysis is mediated by the ubiquitin pathway and requires the action of CDC34, CDC4, CDC53, SKP1, and CLN/CDC28 . As a first step in defining the functions of the corresponding gene products, we have reconstituted SIC1 multiubiquitination in DEAE-fractionated yeast extract . Multiubiquitination depends on cyclin/CDC28 protein kinase and the CDC34 ubiquitin-conjugating enzyme . Ubiquitin chain formation is abrogated in cdc4ts mutant extracts and assembly restored by the addition of exogenous CDC4, suggesting a direct role for this protein in SIC1 multiubiquitination . Deletion analysis of SIC1 indicates that the N-terminal 160 residues are both necessary and sufficient to serve as substrate for CDC34-dependent ubiquitination . The complementary C-terminal segment of SIC1 binds to the S-phase cyclin CLB5, indicating a modular structure for SIC1.

Mol Biol Cell, 1997 Aug, 8(8), 1405 - 14
Protein-protein interactions in the synaptonemal complex; Tarsounas M et al.; In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC) . Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins . We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations . Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions . In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments . The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1 . Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue . The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II.

Biochem J, 1997 Aug 1, 325 ( Pt 3), 587 - 91
A critical amino acid residue, asp446, in UDP-glucuronosyltransferase; Iwano H et al.; An amino acid residue, Asp446, was found to be essential for the enzymic activity of UDP-glucuronosyltransferase (UGT) . We obtained a rat phenol UGT (UGT1*06) cDNA (named Ysh) from male rat liver by reverse-transcription (RT)-PCR using pfu polymerase . A mutant Ysh having two different bases, A1337G and G1384A (named Ysh A1337GC1384A), that result in two amino acid substitutions, D446G and V462M, was obtained by RT-PCR using Taq polymerase . Ysh was expressed functionally in microsomes of Saccharomyces cerevisiae strain AH22 . However, the expressed protein from YshA1337GG1384A had no transferase activity . Two other mutant cDNAs with YshA1337G having one changed base, A1337G, resulting in one amino acid substitution, D446G, and YshG1384A having a changed base, G1384A, resulting in an amino acid substitution, V462M, were constructed and expressed in the yeast . The expressed protein from YshG1384A (named YshV462M) exhibited enzymic activity, but the one from YshA1337G (named YshD446G) did not show any activity at all . Asp446 was conserved in all UGTs and UDP-galactose:ceramide galactosyltransferases reported, suggesting that Asp446 plays a critical role in each enzyme.

Genomics, 1997 Aug 1, 43(3), 307 - 15
A method for genome comparisons and hybridization studies using known megabase-scale DNA sequences as a reference; Qiu P et al.; We present a method for genome comparisons and high-resolution hybridization analyses using megabase stretches of known DNA sequences as a reference . The method employs two-dimensional gel electrophoresis, separating genomic segments cut with different restriction endonucleases in the first and second dimensions, to generate filters suitable for image analysis and repeated nucleic acid hybridizations . The corresponding two-dimensional pattern is computed from the reference nucleotide sequence and matched to the observed pattern, thereby identifying each fragment on the filter; at the same time the technique uncovers discrepancies from the reference sequence . This permits genome comparisons as well as automated identification and quantification of hybridization patterns with various probes . The technique is illustrated by an analysis of Saccharomyces cerevisiae chromosome IX.

J Cell Sci, 1997 Aug, 110 ( Pt 15), 1683 - 92
Cell-type specific calcium signalling in a Drosophila epithelium; Rosay P et al.; Calcium is a ubiquitous second messenger that plays a critical role in both excitable and non-excitable cells . Calcium mobilisation in identified cell types within an intact renal epithelium, the Drosophila melanogaster Malpighian tubule, was studied by GAL4-directed expression of an aequorin transgene . CAP2b, a cardioactive neuropeptide that stimulates fluid secretion by a mechanism involving nitric oxide, causes a rapid, dose-dependent rise in cytosolic calcium in only a single, genetically-defined, set of 77 principal cells in the main (secretory) segment of the tubule . In the absence of external calcium, the CAP2b-induced calcium response is abolished . In Ca2+-free medium, the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, elevates {Ca2+}i only in the smaller stellate cells, suggesting that principal cells do not contain a thapsigargin-sensitive intracellular pool . Assays for epithelial function confirm that calcium entry is essential for CAP2b to induce a physiological response in the whole organ . Furthermore, the data suggest a role for calcium signalling in the modulation of the nitric oxide signalling pathway in this epithelium . The GAL4-targeting system allows general application to studies of cell-signalling and pharmacology that does not rely on invasive or cytotoxic techniques.

Curr Opin Cell Biol, 1997 Aug, 9(4), 573 - 7
Molecular biology of iron and zinc uptake in eukaryotes; Eide D; Recent studies of iron uptake in Saccharomyces cerevisiae have provided insights into the role of multicopper oxidases in eukaryotic metal transport . These studies have also led to the identification of a novel iron transporter in plants and the recognition of a new family of transporter proteins that may participate in metal uptake in a diverse array of eukaryotic speciesPublication Types:

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